'OLOGY 
I3RARY 


THE  UNIVERSITY  OF  CHICAGO 
SCIENCE  SERIES 


Editorial  Committee 
ELIAKIM  HASTINGS  MOORE,  Chairman 

JOHN  MERLE  COULTER 
ROBERT  ANDREWS  MILLIKAN 


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A  CHEMICAL  SIGN  OF  LIFE 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,  ILLINOIS 


Bgents 

THE  BAKER  &  TAYLOR  COMPANY 

NEW  TOBK 

THE  CUNNINGHAM,  CURTISS  &  WELCH  COMPANY 

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THE  CAMBRIDGE  UNIVERSITY  PRESS 

LONDON  AND  EDINBURGH 

THE  MARUZEN-KABUSHIKI-KAISHA 

TOKTO,  OSAKA,    KYOTO,  HJKCOKA,  SENDAI 

THE  MISSION  BOOK  COMPANY 

SHANGHAI 

KARLW.  HIERSEMANN 

LEIPZI8 


A  CHEMICAL  SIGN 
OF  LIFE' 


SHIRO  TASHIRO 

Instructor  in  Physiological  Chcnftstry  in  the  University  of  Chicago 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,  ILLINOIS 


010!  OGY 
tJBP'RY 


COPYRIGHT  1917  BY 
THE  UNIVERSITY  OF  CHICAGO 


All  Rights  Reserved 


Published  March  1917 


Composed  and  Printed  By 

The  University  of  Chicago  Press 

Chicago,  Illinois,  U.S.A. 


PREFACE 

The  present  work  is  an  attempt  to  apply  facts  dis- 
covered during  the  study  of  the  physiology  of  nerves 
to  living  processes  in  general.  That  mechanism  char- 
acteristic of  all  living  matter  which  enables  it  to  respond 
to  the  external  world  is  best  developed  in  the  nervous 
system.  This  mechanism  may  be  called  the  most 
characteristic  thing  in  life.  The  chemical  accompani- 
ment, or  basis,  of  this  mechanism,  discovered  by  the 
author  in  nerve  fibers,  he  has  hoped  to  show  exists 
also  in  all  forms  of  living  matter,  both  of  plants  and  of 
animals.  It  gives  a  chemical  method  of  distinguishing 
living  from  dead  tissue,  and  of  measuring  the  quantity 
of  life. 

This  book,  therefore,  contains  somewhat  in  detail 
all  the  essential  facts  which  .he  with  his  students  has 
discovered  from  studies  of  the  chemical  changes  in 
nerves  accompanying  functional  change.  In  the  pres- 
entation of  this  work,  however,  many  important  refer- 
ences and  discussions  have  been  omitted  in  order  that 
the  reader  may  not  lose  the  main  trend  of  the  argument. 
The  facts  themselves  are  nevertheless  given  in  the  form 
of  accurate  numerical  data  so  that  the  book  may  be 
useful  also  to  the  specialist  whose  interest  lies  more 
directly  in  the  general  physiology  of  the  nervous  system. 

In  an  appendix  the  detailed  method  for  the  use  of  the 
biometer  is  added  in  response  to  frequent  requests  of 
many  friends  and  students  who  wish  to  use  it  for  various 
biological  and  chemical  researches. 

The  author  is  deeply  indebted  to  Professor  A.  P. 
Mathews  for  his  criticism,  scientific  and  literary,  during 

the  preparation  of  the  work. 

SHIRO  TASHIRO 

January,  1917 

34B168 


CONTENTS 

CHAPTER  PAGE 

I.  IRRITABILITY  AS  A  SIGN  OF  LIFE i 

The  Characteristics  of  Living  Matter.  Irritability. 
Functional  Changes  as  a  Sign  of  Irritability.  Functional 
Changes  in  the  Nerve  Fiber. 

II.  CHEMICAL  SIGNS  OF  IRRITABILITY  IN  THE  NERVE 
FIBER     . 10 

Signs  of  Metabolic  Activities  in  a  Tissue.  Indirect  Evi- 
dence for  Presence  of  Metabolic  Activity  in  the  Nerve 
Fiber.  Direct  Evidence.  Experimental  Methods  with  Non- 
Medullated  Nerve  Fiber.  Medullated  Nerve  Fiber.  Is 
Carbon  Dioxide  Produced  by  Living  Processes  ?  Discussion. 

III.  CHEMICAL  SIGNS  OF  IRRITABILITY  IN  THE  NERVE 
FIBER — Continued 34 

Increased  Metabolism  on  Stimulation.  Experimental. 
Electrical  Stimulation.  Other  Stimulations.  Discussion. 

IV.  EXCITATION  AND  CONDUCTION 57 

Excitability  and  Metabolism.  Degree  of  Excitability  Com- 
pared with  Metabolism.  Effects  of  Narcotics.  Lower 
Concentration.  Higher  Concentration.  Direction  of  the 
Nerve  Impulse  and  Metabolic  Gradient.  Velocity  of  the 
Nerve  Impulse  and  Carbon  Dioxide  Production. 

V.  CHEMICAL  SIGNS  OF  LIFE 87 

Resting  Metabolism  in  Seeds.  Increased  Metabolism  in 
Seeds  on  Injury.  Other  Tissues.  A  Chemical  Sign  of  Life. 

VI.  CONCLUSIONS 95 

Summary.  Other  Criteria  of  Life.  Functions  of  Resting 
Metabolism.  Metabolism  and  Irritability.  Chemical  Con- 
ditions in  the  Living  Processes.  Quantity  of  Life. 

APPENDIX.    THE  BIOMETER:  How  TO  USE  IT    .     .     .109 

ix 


CHAPTER  I 

IRRITABILITY  AS  A  SIGN  OF  LIFE 

In  the  pages  which  follow  we  shall  consider  par- 
ticularly the  question  of  the  chemical  processes  which 
take  place  in  nerves  when  nerve  impulses  pass  over 
them.  There  is  scarcely  a  subject  in  the  world  more 
interesting  than  this  one,  for  the  question  of  what  is 
the  nature  of  that  disturbance  in  nerve  tissue  which 
shows  itself  in  our  thoughts  has  attracted  men  from  the 
earliest  days.  We  must  first  find  out  the  changes 
of  a  material  kind — if  there  are  any  such  changes— 
which  occur  in  the  brain  when  we  think  before  we  can 
form  any  probable  idea  of  the  relation  of  these  changes 
to  the  psychical  changes  which  accompany  them. 
Obviously,  we  must  first  try  to  solve  the  simplest  prob- 
lem in  this  field  and  discover  what  are  the  changes 
of  a  chemical  or  physical  kind  when  a  nerve  impulse 
flashes  over  a  nerve  before  we  can  form  any  conception 
of  the  relation  of  the  material  to  the  psychic  world. 
The  following  pages  do  not  contain,  of  course,  the  solu- 
tion of  this  problem  of  such  absorbing  interest,  but 
they  do  present  the  first  accurate  information  we  have 
had  of  the  chemical  changes  which  accompany  the 
nerve  impulse;  they  have  in  them,  therefore,  the 
foundation  upon  which  a  solid  structure  of  fact  can  be 
based. 

.    The  observations  which  we  have  to  present  are  not 
confined  to  nerves,  however,  for  psychic  phenomena  are 


2        ;  \  CHEMICAL  SIGN  OF  LIFE 

not  confined,  we  believe,  to  animals  which  have  well- 
developed  nerves.  The  course  of  evolution  from  the 
simplest  to  the  most  complex  shows  us  very  clearly  that 
the  complex  psychic  life  of  man  and  the  higher  animals 
did  not  suddenly  spring  fully  formed  into  existence.  In 
every  child,  in  fact,  it  can  be  seen  to  appear  very  slowly 
and  gradually  and  to  increase  as  the  child  develops.  We 
cannot  say  at  what  point  psychic  life  begins,  for  the 
simplest  organisms  show  some  signs  of  it.  Indeed,  as 
living  originates  from  lifeless,  we  are  led  to  conclude 
that  the  simplest  rudiments  of  psychic  life  must 
be  found  also  in  the  lifeless.  And  perhaps  the  universe 
as  a  whole,  inert  as  it  appears  to  us  to  be,  may  have  a 
f  psychic  life  of  its  own.  So  it  is  not  necessary  to  con- 
fine our  studies  to  nerves,  for  we  find  the  same  phe- 
nomena which  nerves  show,  phenomena  corresponding  to 
those  of  nerve  impulses,  even  in  plants,  and  indeed  in 
the  simplest  kinds  of  plants.  The  differences  between 
animals  and  plants  are  superficial  differences.  Plants, 
in  general,  are  sessile;  they  cannot  move  freely  from 
place  to  place  as  animals  do;  and  they  have  a  green 
pigment  in  them — chlorophyll — while  most  animals 
have  a  red  pigment  in  their  blood.  This  green  pigment 
enables  plants  to  make  their  food  from  simpler  substances 
than  can  be  done  by  animals.  But  these  differences 
are  superficial,  and  fundamentally  plants  and  animals 
are  alike.  We  must  suppose,  therefore,  that  even  so 
humble  a  living  form  as  a  small  plant  seed  has  a  psychic 
life  of  its  own.  Impulses  pass  through  it  like  nerve 
impulses;  it  may  be  anesthetized  as  in  the  case  of 
man;  it  sleeps  as  does  man;  and,  indeed,  many  of  the 
fundamental  properties  it  shows  resemble  those  which 


IRRITABILITY  AS  A  SIGN  OF  LIFE  3 

we  possess.  Thus  a  part  of  the  study  which  follows  has 
been  made  upon  such  simple  things  as  seeds  and  garden 
peas.  It  is  surprising  how  closely  the  results  obtained 
with  these  parallel  those  obtained  from  nerves. 

The  really  important  and  peculiar  property  of  living 
things  is  the  psychic  life  they  show.  And  if  we  actually 
had  an  accurate  means  of  testing  the  degree  or  amount 
of  life,  it  would  be  some  kind  of  a  reagent  or  instrument 
for  testing  "psychism,"  as  we  may  call  it.  But  un- 
fortunately we  cannot  at  present  find  any  means  of  test- 
ing this  property.  We  do  not  know  what  its  physical 
basis  is,  and,  until  we  discover  that,  we  cannot  make 
a  psychometer  which  we  can  apply  to  all  kinds  of 
living  and  non-living  things,  and  thus  measure  the 
amount  of  psychism,  and  hence  of  life,  which  they 
possess.  In  the  absence  of  any  such  psychometer  we 
have  to  do  the  best  we  can,  and  take  as  a  measure  of 
this  property  those  physical  and  chemical  changes  which 
experience  or  experiment  demonstrates  to  us  always 
accompany  the  psychic  change.  The  situation  is  very 
,  much  as  it  was  in  the  realm  of  electricity  before  the 
.  galvanometer  was  invented;  an  idea  of  the  quantity 
!  of  electricity  produced  by  a  battery  could  be  obtained 
:  only  indirectly  by  measuring  the  amount  of  chemical 
change  which  the  current  produced,  since  Faraday 
found  that  that  amount  was  always  a  measure  of  the 
amount  of  electricity. 

There  are  material  changes  which  occur  in  living 
things  as  long  as  they  are  alive  and  show  psychic  life 
of  any  kind.  The  changes  which  we  may  rely  upon  to 
measure  the  amount  of  life,  and  thus  indirectly  the 
amount  of  psychism,  are  partly  visible  changes,  but  in 


4  A  CHEMICAL  SIGN  OF  LIFE 

part  they  are  invisible  and  have  to  be  detected  by  special 
apparatus.  The  visible  changes  consist  in  some  reaction 
of  the  organism  when  its  surroundings  change.  If  it 
moves  when  it  is  touched,  the  degree  of  movement  not 
being  related  to  the  impulse  given  it;  if  it  breathes;  if 
it  changes  its  rate  of  growth  under  changing  conditions, 
we  say  that  it  is  alive.  It  is  irritable,  and  it  has  the 
property  of  irritability.  Another  thing  noticed  in  a 
living  thing  is  that  the  impulse  which  arouses  it  to 
action  may  cause  reaction  in  a  part  of  the  organism 
distant  from  the  point  of  stimulation.  In  other  words, 
the  change,  whatever  its  nature,  set  up  in  the  organism 
by  the  stimulus  is  propagated  to  a  distance  from  the 
point  of  irritation.  We  can  see  the  results  of  this  propa- 
gation. All  things  which  show  this  conduction  and 
response  we  say  are"  living  things.  These  are  physical 
processes  which  apparently  always  accompany  the 
psychic  process.  But  sometimes  these  changes,  although 
they  occur,  produce  no  visible  result;  consequently  we 
must  have  methods  which  will  detect  conduction  and 
irritability,  even  though  there  are  no  visible  signs  of 
them.  One  cannot  see,  for  example,  that  anything  has 
happened  to  a  seed  when  it  is  pricked  by  a  pin;  it 
does  not  say  "ouch!"  loud  enough  for  us  to  hear  it,  or 
in  a  language  we  understand;  but  nevertheless  it  jumps 
when  it  is  pricked  as  if  it  did  say  "ouch!"  as  we  can 
show  by  appropriate  methods. 

There  are  two  signs,  or  tests,  which  all  living  things 
show  and  which  are  an  index  of  life.  One  of  these  is  an 
electrical  disturbance.  This  was  discovered  a  very  long 
time — a  hundred  years — ago,  and  its  discovery  was  the 
basis  of  the  development  of  knowledge  of  electricity.  The 


IRRITABILITY  AS  A  SIGN  OF  LIFE  5 

other  is  a  chemical  sign,  which  has  just  been  discovered 
and  which  will  be  discussed  in  this  book.  The  electrical 
sign  of  life  was  discovered  by  Galvani  when  he  found  that 
animal  tissues  are  a  source  of  electricity.  He  discovered 
animal  electricity.  It  is  now  certain  that-  whenever 
the  response  to  a  stimulus  takes  place  in  animals  or 
plants — the  response  which  is  the  sign  of  life — an  elec- 
trical change  accompanies  it.  By  placing  a  galvanome- 
ter on  the  animal  or  plant  we  can  study  this  electrical 
response.  Life  and  electricity  are  thus  shown  to  be 
related.  Electricity  and  psychism  have  something  in 
common,  although  just  what  the  connection  is  cannot  at 
present  be  said.  The  English  physiologist  Waller  has 
recently  introduced  as  a  measure  of  life  a  particular  kind 
of  electrical  response  which  he  has  discovered  and  which 
he  calls  the  " blaze"  current,  because  it  is  as  if  the 
electrical  display  suddenly  blazed  up  when  the  living 
matter  was  disturbed;  this  he  calls  an  electrical  sign  of 
life.  By  it  he  can  tell  whether  a  dry  seed  is  alive  or  not 
without  putting  it  in  the  ground  and  letting  it  sprout. 
It  is  very  hard  to  know  whether  this  electrical  dis- 
turbance which  living  things  show  is  due  to  physical  or 
to  chemical  changes  in  their  substances. 

It  is  therefore  a  matter  of  very  great  interest  that 
I  have  recently  found  that  there  is  always  and  every- 
where an  accompanying  chemical  change  of  a  particular 
kind  which  is  as  sure  a  sign  of  life  and  as  invariable  an 
accompaniment  of  the  vital  reaction  as  the  electrical 
change.  This  chemical  sign  is  the  sudden  outburst  of 
carbon  dioxide  which  all  living  things  show — plants  as 
well  as  animals,  dry  seeds  as  well  as  the  nerve  tissues  of 
the  highest  mammals — when  they  are  stimulated  in  any 


6  A  CHEMICAL  SIGN  OF  LIFE 

way.  The  instrument  which  I  have  made  to  detect 
this  carbon  dioxide  I  have  called  a  "biometer"  because, 
as  will  be  appreciated  from  this  short  discussion,  it  is 
an  apparatus  for  measuring  or  detecting  the  amount  of 
life  possessed  by  different  things.  I  shall  show  in  the 
following  pages  that  the  increment  of  carbon  dioxide 
produced  by  living  things  when  they  are  irritated,  or 
stimulated  in  any  way,  is  a  sure  measure  of  the  amount 
of  life  they  have;  and  we  may  hope  that  it  is  to  be  an 
indirect  measure  of  the  amount  of  psychism  they  possess, 
although  of  course  we  cannot  be  sure  of  this  as  yet.  It 
will  be  noticed  that  it  is  not  the  absolute  amount  of 
carbon  dioxide  which  is  the  measure  of  life,  but  the 
increase  above  the  usual  production  which  occurs  when 
a  definite  amount  of  stimulus  is  applied  to  the  living 
thing,  which  is  the  real  measure  of  life.  Anesthetized 
or  sick  things  do  not  show  the  normal  increase;  those 
abounding  in  life  show  a  remarkable  increase. 

The  first  results  to  be  presented  will  be  the  proof  that 
carbon  dioxide  production  is  the  sign  of  life  of  a  nerve 
fiber.  And  it  will  be  well  before  going  into  this  to  say  a 
few  words  about  the  scientific  opinion  concerning  the 
nature  of  the  nerve  impulse  generally  prevailing  before 
the  work  recorded  here  was  done. 

The  main  function  of  a  nerve  fiber  is  to  transmit  a 
state  of  excitation  from  one  place  to  another.  It  serves 
for  the  conduction  of  the  nerve  impulse,  which  it  trans- 
mits in  the  most  efficient  manner.  The  nerve  is  also 
excitable  at  all  points,  since  it  can  be  stimulated  by  a 
variety  of  methods  at  any  point  along  the  fiber.  When 
physiologists  investigated  what  takes  place  in  nerve 
fibers  during  the  passage  of  nerve  impulses,  many 


IRRITABILITY  AS  A  SIGN  OF  LIFE  7 

peculiar  results  were  brought  out.  In  the  first  place, 
if  there  are  no  other  organs  attached  to  the  nerve  it  is 
impossible  to  determine  by  casual  observation  whether 
or  not  the  nerve  has  been  stimulated,  for  there  is  posi- 
tively no  visible  physical  sign  of  the  vitality  in  it/  Not 
even  with  a  microscope  can  any  structural  change  in  the 
tissue  be  seen.  There  is,  also,  no  heat  change  detected 
with  a  method  which  is  sensitive  to  a  millionth  of  a 
degree  Centigrade.  There  was,  before  this  work  was 
published,  no  apparent  production  of  carbon  dioxide, 
or  any  other  chemical  change  in  the  tissue.  These  facts 
seemed  to  indicate  that  the  functional  activity  of  nerve 
fibers  was  in  no  way  associated  with  any  chemical 
change.  This  failure  of  a  nerve  to  show  any  chemical 
or  structural  changes  similar  to  those  of  muscles  had  a 
decisive  influence  in  the  formation  of  ideas  concerning, 
not  only  the  nature  of  the  nerve  impulse,  but  also  the 
nature  of  irritability  in  general.  For  nerve  fibers 
not  only  show  the  highest  type  of  irritability  of  proto- 
plasm, but  they  also  possess,  as  stated  before,  the  power 
of  transmitting  the  state  of  excitation  in  the  most  perfect 
manner.  And  all  attempts  to  explain  the  nature  of 
irritability  in  general  must  necessarily  account  for  the 
peculiarities  of  the  nerve  fiber  where  we  find  that  prop- 
erty in  its  highest  development.  If  irritability,  excita- 
tion, and  conduction  do  not  involve  chemical  changes 
in  nerves,  it  may  be  concluded  that  neither  do  they  in 
any  other  tissues.  Thus,  on  account  of  the  absence 
of  evidence  of  any  chemical  changes  accompanying  irrita- 
bility in  nerves,  we  have  gradually  drifted  away  from  the 
notion  that  the  fundamental  condition  for  protoplasmic 
activity  is  chemical. 


A  CHEMICAL  SIGN  OF  LIFE 

When  it  was  found  that  an  electrical  change  occurred 
in  a  nerve  when  it  conducted  an  impulse,  the  problem  was 
considered  to  be  settled.  The  nerve  impulse  was  sup- 
posed to  be  electrical  in  nature.  This  idea  was  soon 
questioned,  however,  when  the  speed  of  the  conduction 
of  a  nerve  impulse  was  found  to  be  so  slow  in  comparison 
with  that  of  an  electrical  current.  The  speediest  nerves, 
such  as  those  of  human  beings,  conduct  impulses  only 
at  the  rate  of  a  hundred  meters  per  second,  whereas 
electricity  travels  in  a  wire  at  a  speed  of  thousands  of 
kilometers  per  second.  One  thing  seemed  to  be  certain — 
that  the  nerve  impulse  can  pass  through  a  fiber  without 
consuming  any  material.  It  was  found  that  some 
nerves  could  not  be  fatigued  even  on  prolonged  stimula- 
tion. This  fact  supported  the  idea  that  certain  quickly 
reversible  physical  conditions  must  exist  in  the  nerve,  and 
that  the  changes  in  these  conditions,  rather  than  chemical 
changes,  must  determine  the  phenomena  of  irritability 
and  conductivity.  Ultimately  physiologists  settled 
down  to  the  view  that  the  physical  and  fundamental 
changes  concerned  in  irritability  were  either  a  change 
of  colloidal  state,  of  surface  tension,  or  in  the  permea- 
bility of  the  nerve  to  salt,  or  changes  in  the  distribution 
of  electrically  charged  particles  in  the  nerve. 

Although  such  physical  changes  as  these  in  nerves 
have  never  been  demonstrated  experimentally,  biolo- 
gists generally  have  tried  to  explain  the  nature  of  a 
nerve  impulse  and  the  phenomena  of  excitation  purely 
on  the  basis  of  these  hypothetical  physical  changes; 
and  they  have  neglected  the  chemical  changes.  They 
have  also  attributed  many  other  important  physiological 
functions,  such  as  secretion  and  contractility,  to  these 


IRRITABILITY  AS  A  SIGN  OF  LIFE  9 

purely  physical  conditions,  and  this  in  spite  of  the  fact 
that  this  point  of  view  is  obviously  incomplete,  if  not 
fundamentally  erroneous,  because  the  source  of  all 
energy  of  living  things  is  chemical.  The  chief  reason, 
as  has  been  stated,  for  the  adoption  of  this  hypothesis 
was  the  fact  that  the  most  irritable  tissue — nervous 
tissue — had  shown  no  sign  of  chemical  changes  when  it 
functioned,  and  also  the  fact  that  some  other  living 
tissues,  such  as  seeds,  seemed  to  maintain  their  vitality 
without  any  chemical  change.  The  erroneous  character 
of  this  view  will  be  apparent  from  what  follows,  where 
it  is  shown  that  chemical  changes  are,  indeed,  an  inva- 
riable accompaniment  of  nervous  activity,  and  of  all  life. 


CHAPTER  II 

CHEMICAL  SIGNS  OF  IRRITABILITY  IN  THE 
NERVE  FIBER 

There  are  various  chemical  processes  which  occur  in 
all  forms  of  living  matter  and  which  we  might  examine 
in  order  to  see  whether  they  are  associated  with  the 
property  of  irritability,  but  we  naturally  seek  to  make 
use  of  that  one  which  is  the  easiest  to  detect.  Among 
these  chemical  processes  there  are,  in  the  first  place, 
the  processes  concerned  in  growth.  All  living  matter 
has  the  power  of  building  up  complex  proteins,  fats, 
and  carbohydrates  as  long  as  it  is  vigorously  alive. 
But  it  is  clear  that  this  process  would  be  very  hard  to 
measure  quantitatively  without  killing  the  living  matter 
and  determining  how  much  substance  it  has  produced. 
And  there  are  also  other  objections  to  using  growth  as  a 
measure  of  vitality.  Another  chemical  process  found 
in  all,  or  nearly  all,  forms  of  matter  is  respiration.  By 
respiration  we  mean  the  gaseous  exchange  of  living 
matter  with  its  environment:  the  taking  on  of  oxygen 
and  the  production  of  carbon  dioxide.  This  is  a  very 
much  more  promising  line  of  experiment  to  follow  in 
measuring  life  and  metabolism,  for,  in  the  first  place,  it 
is  universal,  as  I  shall  presently  show,  and,  in  the  second 
place,  the  oxygen  may  be  measured,  or  the  carbon 
dioxide  given  off  may  be  determined,  without  injuring 
the  living  matter.  It  was  for  this  reason  that  the 
carbon  dioxide  was  selected  for  study  as  probably 


CHEMICAL  SIGNS  OF  IRRITABILITY  n 

being  the  easiest  of  quantitative  determination  and 
undoubtedly  correlated  with  the  most  fundamental 
vital  processes. 

The  idea  that  respiration  is  one  of  the  most  funda- 
mental of  vital  phenomena  is  by  no  means  a  novel  view. 
Even  in  the  earliest  times  breathing  was  supposed  to 
be  the  process  most  intimately  connected  with  life. 
When  a  man  stopped  breathing  he  died.  As  early  as 
the  second  century  Galen  had  the  notion  that  there  must 
be  a  pneumatic  spirit  in  the  air  which  kept  up  life, 
and  he  predicted  that  some  day  it  would  be  discovered. 
It  was  after  the  lapse  of  fifteen  hundred  years  that  this 
prediction  was  verified  or  fulfilled  when  Mayow,  an 
English  physician,  discovered  that  there  was  a  gas,  or 
spirit,  in  the  air  which  was  essential  to  life  and  com- 
bustion. Later,  oxygen  was  discovered  by  Priestly,  and 
it  was  Lavoisier  who  first  showed  that  this  oxygen  after 
entering  the  lungs  came  out  again  as  carbon  dioxide; 
and  he  proved  that  animal  heat  was  due  to  the  com- 
bustion of  the  materials  of  the  body  by  the  oxygen  to 
form  water  and  carbon  dioxide,  and  that  the  sole  source 
of  energy  of  living  things  was  this  combustive  change. 
In  selecting  respiration  as  the  chemical  test  of  life  we 
are,  therefore,  selecting  that  most  fundamental  reaction 
by  virtue  of  which  living  things  get  their  energy.  It 
is  clear  that  it  is  this  reaction,  rather  than  any  other 
chemical  reaction,  which  touches  most  closely  the 
phenomena  of  irritability;  for,  to  move  or  to  think, 
we  must  have  energy.  It  is  much  better  to  take  this 
reaction,  rather  than  those  chemical  changes  which 
are  related  to  growth  or  the  repair  of  waste,  as  a 
criterion  of  living,  for  the  very  essence  of  a  living  thing 


12  A  CHEMICAL  SIGN  OF  LIFE 

is  that  by  chemical  transformations  it  sets  free  energy 
and  moves  itself. 

It  is  much  better,  too,  to  take  the  carbon  dioxide  pro- 
duced, rather  than  the  oxygen  consumed,  as  the  measure 
of  the  metabolism  associated  with  irritability,  for  the 
reason  that  sometimes  organisms  get  their  oxygen  from 
sources  other  than  the  air,  whereas  their  carbon  dioxide 
production  is  always  something  positive  and  universal. 

Indirect  evidence  of  the  presence  of  metabolic  activity 
in  the  nerve  fiber. — The  search  for  some  kind  of  metab- 
olism, such  as  the  production  of  carbon  dioxide,  in 
nerves  had  been  made  by  many  physiologists  on  many 
occasions,  but  it  was  impossible  for  them  to  discover 
this  substance  because  their  methods  were  not  sufficiently 
delicate.  No  carbon  dioxide  could  be  found,  and  for 
this  and  other  reasons  the  conclusion  was  incorrectly 
drawn  that  there  was  none  produced,  or  that,  if  it  were 
produced,  it  had  no  connection  with  the  vital  functions 
of  the  nerve.  Most  physiologists  were  accordingly  of  the 
opinion  that  the  conduction  of  the  nerve  impulse  was 
a  physical  process  and  involved  no  transformation  of 
energy  and  no  consumption  of  material.  There  was 
one  exception  to  this  rule.  Professor  A.  D.  Waller, 
the  eminent  English  physiologist,  maintained  that, 
because  of  their  electrical  behavior,,  nerves'  certainly 
produced  carbon  dioxide.  In  1896  he  showed  that 
carbon  dioxide  when  applied  to  a  nerve  produced  a  very 
characteristic  change  in  the  electrical  response  which  a 
nerve  exhibits  when  it  is  irritated.  It  will  be  remem- 
bered that  when  a  nerve  or,  in  fact,  any  kind  of  proto- 
plasm is  irritated  in  any  way,  if  one  applies  two  electrodes 
to  the  living  tissue  in  such  a  way  that  one  electrode  is  on 


CHEMICAL  SIGNS  OF  IRRITABILITY  13 

a  part  which  is  in  activity  and  the  other  on  a  part  which 
is  less  active,  it  will  be  found  that  there  is  a  current 
which  flows  in  the  tissue  from  the  more  active  to  the  less 
active  part,  from  the  more  to  the  less  excited,  and  outside 
in  the  galvanometer  from  the  less  to  the  more  excited 
part.  This  electrical  current  was  discovered  by  Galvani 
and  is  called  the  current  of  action,  or  the  action  current. 
Generally,  when  an  impulse  sweeps  along  a  nerve  to 
which  two  electrodes  are  applied,  first  one  electrode 
and  then  the  other  becomes  negative,  so  that  the  current 
is  diphasic,  running  first  in  one  direction  and  then  in  the 
other.  Now,  Waller  observed  that  when  a  nerve  was 
exposed  to  carbon  dioxide  this  diphasic  current  showed 
a  characteristic  change,  the  negative  phase  being  first 
increased  by  small  amounts  of  carbon  dioxide  and  then 
diminished.  He  then  discovered  that  just  the  same  kind 
of  a  change  occurred  in  the  electrical  response  if  he  stimu- 
lated a  nerve  repeatedly  at  very  short  intervals  of  time. 
He  concluded  from  this  that  on  stimulation  of  the  nerve 
carbon  dioxide  was  produced,  and  that  this  caused  the 
characteristic  alteration  of  the  electrical  response  which 
occurred  in  the  tetanized  or  repeatedly  stimulated  nerve. 
This  conclusion  was  not  generally  accepted  by  physiol- 
ogists for  the  reason  that  it  was  possible  that  the  same 
change  in  the  electrical  response  might  be  produced  in 
other  ways  than  by  carbon  dioxide,  and  while  the  experi- 
ments were  regarded  as  circumstantial  evidence  of  value, 
showing  that  a  chemical  change  accompanied  the  nerve 
impulse,  they  were  not  regarded  as  conclusive. 

Waller  supported  this  conclusion  by  another  dis- 
covery, namely,  that  when  he  stimulated  the  nerve  at 
regular  intervals,  not  too  long  or  too  short,  by  a  strong 


14  A  CHEMICAL  SIGN  OF  LIFE 

induction  shock,  he  obtained  a  series  of  electrical 
responses,  each  of  which  is  the  greatest  the  nerve  can 
give  at  the  time,  but  each  of  which  is  a  little  greater 
than  its  predecessor.  Such  a  series  of  increasing  re- 
sponses is  known  as  a  staircase,  the  negative  phase 
increasing  steadily  while  the  positive  phase  decreases. 
This  Waller  explained  by  supposing  that  small  amounts 
of  carbon  dioxide  were  formed  by  each  nerve  activity, 
and  that  this  augmented  the  negative  response  and 
diminished  the  positive  response,  just  as  does  carbon 
dioxide  applied  to  the  outside  of  the  fiber.  He  considered 
that  our  failure  to  find  the  gas  was  due  to  the  inadequacy 
of  the  chemical  methods  then  in  existence.  That  this 
criticism  of  Waller's  was  a  just  one  and  that  there  may 
be  carbon  dioxide  produced  by  nerves,  but  too  small  in 
amount  to  be  measured  by  the  ordinary  chemical  method, 
is  shown  by  the  following  calculation:  A  frog  (Rana 
temporarid)  gives  off  0.355  g-  °f  carbon  dioxide  per 
kilogram  per  hour  at  19°  to  20°  C.  A  small  piece  of 
the  nerve  fiber  of  the  same  animal,  say  i  cm.,  or  three- 
eighths  of  an  inch,  in  length,  will  weigh,  probably,  not 
more  than  10  mg.  Now,  if  this  mass  of  the  nerve 
fiber  respires  at  the  same  rate  as  the  whole  animal,  it 
will  not  give  off  more  than  about  0.000,000,7  g-  °f 
carbon  dioxide  during  ten  minutes.  This  calculation 
at  once  suggested  that  the  failure  to  detect  the  evolution 
of  carbon  dioxide  in  nerves  was  very  probably  due  to  the 
limitation  of  the  methods  for  the  estimation  of  the  carbon 
dioxide,  and  that  it  was  not  at  all  conclusive  evidence 
that  carbon  dioxide  was  not  produced.  It  was  evidently 
necessary  to  devise  methods  for  the  detection  of  very 
minute  quantities  of  carbon  dioxide. 


CHEMICAL  SIGNS  OF  IRRITABILITY  15 

'  In  order  to  study  the  whole  of  the  respiratory  metab- 
olism of  a  tissue,  we  should  at  least  determine  the 
oxygen  consumption  as  well  as  the  carbon  dioxide 
production,  and  also  generally  the  heat  production. 
Inasmuch  as  the  present  problem,  however,  is  concerned 
only  with  presenting  direct  evidence  for  the  existence 
of  metabolic  activity  in  nerve  fibers,  we  shall  attempt 
to  measure  the  carbon  dioxide  production  alone;  for 
while  the  lack  of  consumption  of  atmospheric  oxygen 
may  not  necessarily  indicate  the  absence  of  chemical 
changes,  the  production  of  carbon  dioxide  will  surely 
prove  the  presence  of  metabolism,  provided,  of  course, 
that  we  can  prove  that  such  carbon  dioxide  is  formed  by 
physiological  processes.  Furthermore,  as  carbon  dioxide 
is  the  only  universal  expression  of  the  respiratory^ 
activity  in  almost  all  anaerobic  and  aerobic  plant  and 
animal  tissues  in  normal  condition,  metabolic  activity 
is  probably  better  represented  by  carbon  dioxide  pro- 
duction than  by  oxygen  consumption,  although  we 
must  restate  here,  of  course,  that  the  study  of  carbon 
dioxide  alone  will  never  reveal  completely  the  nature  of 
the  metabolic  activity. 

Method. — The  method  which  was  finally  devised  to 
detect  and  measure  quantitatively  the  very  minute 
amounts  of  carbon  dioxide  which  it  might  be  expected* 
would  be  formed  consisted  essentially  in  determining 
the  amount  of  carbon  dioxide  which  was  just  sufficient 
to  produce  a  deposit  of  barium  carbonate  in  a  film  of 
half-saturated  barium  hydroxide  solution.  Barium  car- 
bonate is  almost  entirely  insoluble  in  such  a  barium 
hydroxide  solution,  and  a  very  small  amount  of  precipi- 
tate can  be  detected  with  the  aid  of  a  small  lens.  The 


1 6  A  CHEMICAL  SIGN  OF  LIFE 

method  is  described  in  detail  in  the  Appendix.  The 
special  apparatus,  the  biometer,  as  I  have  named  it, 
which  was  constructed  for  the  investigation,  is  shown  in 
Fig.  i,  and  its  use  is  detailed  in  the  Appendix.  It  will 
detect  one  ten-millionth  of  a  gram  of  carbon  dioxide  and 
estimate  it  with  accuracy. 

As  is  shown  in  the  figure,  the  biometer  has  two 
respiratory  chambers  each  provided  with  a  small  tube,  at 
the  top  of  which  the  hemispherical  drop  of  barium 
hydroxide  can  be  formed.  Exceedingly  minute  amounts 
of  carbon  dioxide  produced  in  the  chamber  by  the  small 
piece  of  nerve  will  be  precipitated  as  barium  carbonate 
on  the  surface  film  of  these  hemispherical  drops  and  may 
be  seen  with  a  lens.  As  the  apparatus  has  two  chambers, 
not  only  can  we  detect  very  small  amounts  of  carbon 
dioxide  which  the  nerve  may  produce,  but  we  can  also 
compare  the  output  of  carbon  dioxide  of  different 
tissues  under  the  same  conditions,  by  placing  one 
tissue  in  one  respiratory  chamber  and  another  in  the 
other. 

To  discover  whether  nerve  fibers,  as  distinct  from 
nerve  cells,  respire,  particular  care  was  taken  to  select 
at  first  those  nerves  which  are  known  to  be  free  from 
such  cells  and,  as  far  as  possible,  free  also  from  connective 
tissue.  It  was  necessary  to  do  this  because  the  work  of 
several  investigators  seemed  to  indicate  that  tissue  oxi- 
dation was  in  some  way  dependent  on  the  cell  nucleus. 
Certain  biologists  even  went  so  far  as  to  believe  that  a 
nerve  fiber  ought  not  to  respire  at  all,  since  it  contained 
no  nucleus.  The  fact  that  the  blood  supply  to  the 
brain,  where  most  of  the  nerve  cells  are  located,  is  so 
copious,  whereas  the  blood  supply  to  the  nerve  fibers  is 


CHEMICAL  SIGNS  OF  IRRITABILITY  17 


FIG.  i. — The  Biometer.     One-fourth  actual  size 


s 


1 8  A  CHEMICAL  SIGN  OF  LIFE 

so  scanty,  was  partly  responsible  for  this  conclusion.1 
In  order  to  test  the  correctness  of  such  an  assumption, 
we  have  studied  quantitatively  the  output  of  carbon 
dioxide  from  various  lengths  of  nerves  which  are  known 
to  be  free  from  nerve  cells  and  which  have  almost  no 
connective  tissue  in  them.  For  this  purpose  the  claw 
nerve  from  the  spider  crab  was  selected. 

Nerve  fibers  are  of  two'  kinds,  called  respectively 
medulla  ted  and  non-medulla  ted  nerves.  The  essential 
conducting  parts  of  these  are  alike,  but  the  medullated 
fibers  have  lying  about  the  conducting  core  of  the  fiber  a 
white,  glistening,  fatty  matter  called  the  medulla,  or 
myelin  sheath.  Most  of  the  nerves  going  to  voluntary 
muscle  in  the  higher  vertebrates  are  medullated;  but 
the  nerves  to  the  viscera  are  often  non-medullated 
and  the  nerves  of  the  invertebrates  are  usually  non- 
medullated.  _  This  medullary  sheath  is  evidently  some- 
thing which  is  found  in  those  nerves  which  it  is  important 
should  conduct  very  quickly  and  which  should  not  be 
fatigued  by  conduction,  and  it  is  clear  that  the  medulla- 
tion  is  an  improvement  which  has  not  yet  been  universally 
introduced.  The  function  of  this  sheath  is  probably 
nutritive.  But  in  any  case  it  is  important,  if  we  wish  to 
avoid  any  complication  which  it  may  introduce  into  the 
physiology  of  the  nerve,  to  examine  both  medullated  and 
non-medullated  nerves.  And  that  we  have  done. 

Non-medullated  nerve  fibers. — When  an  isolated  claw 
nerve  of  the  spider  crab  is  placed  in  the  right  chamber 

1  Indeed,  Bayliss  attributes  our  results,  which  are  soon  to  be  detailed, 
to  the  presence  of  the  connective  tissue  cells  around  the  fiber,  so  firmly 
convinced  does  he  appear  to  be  that  only  nucleated  parts  of  cells  respire. 
For  further  consideration  of  this  objection  see  p.  33. 


CHEMICAL  SIGNS  OF  IRRITABILITY  19 

of 'the  biometer  and  no  nerve  in  the  left,  the  biometer 
being  properly  sealed  with  mercury  and  filled  with  air 
which  is  free  from  carbon  dioxide,  and  if  barium  hydrox- 
ide is  allowed  to  rise  to  the  top  of  each  tube  in  such  a 
way  as  to  form  hemispherical  drops  of  approximately 
equal  size  in  both  chambers,  we  observe  that  the  drop 
in  the  right  chamber,  where  the  nerve  is,  will  soon  be 
coated  with  a  white  precipitate  of  barium  carbonate, 
but  that  no  precipitate  whatever  can  be  seen,  even  with 
a  lens,  in  the  left  chamber.  Carbon  dioxide  is  thus 
shown  to  be  produced  by  this  resting  nerve  of  the  spider 
crab.  By  interchanging  the  nerve  from  the  right  to  the 
left  chamber,  no  nerve  being  now  put  in  the  right,  we 
find  that  the  precipitate  is  now  in  the  left-hand  side  of 
the  biometer,  and  we  have  no  difficulty  in  convincing 
ourselves  that  the  carbon  dioxide  has  come  from  the 
nerve,  for  we  have  thus  eliminated  any  technical  error 
which  might  have  produced  the  different  results  in  the 
different  chambers.  The  rate  at  which  the  precipitate 
appears  and  its  quantity  depend  on  the  size  of  the  nerve 
and  the  length  of  time  we  leave  it  in  the  chamber.  That 
an  unstimulated  nerve  gives  off  carbon  dioxide  is  a  fact 
which  can  thus  be  demonstrated  easily  to  anyone  if  the 
proper  apparatus  is  at  hand.  The  rate  of  production 
of  carbon  dioxide  by  the  normal  resting  nerve  of  the 
spider  crab  is  found  to  be  proportional  to  its  weight, 
other  things  being  equal,  and  is  fairly  constant.  The 
quantitative  determination  shows  that  for  10  mg.  of 
nerve  per  ten  minutes  it  gives  off  6.  7 X  lo""7  g.  of  carbon 
dioxide  at  15°  to  16°  C. 

The  quantitative  determination  of  this  amount  is  made  in 
the  following  manner:  The  claws  of  t-he  crab  are  carefully  removed 


20  A  CHEMICAL  SIGN  OF  LIFE 

from  the  body,  and  by  gentle  cracking  the  long  fiber  of  the  nerve 
trunk  is  easily  isolated.  After  the  last  drop  of  water  is  removed 
by  a  filter  paper,  the  nerve,  with  the  aid  of  glass  chopsticks,  is 
carefully  placed  on  the  glass  plate  (Fig.  2)  and  quickly  weighed. 
The  glass  plate  with  the  nerve  is  now  hung  on  the  platinum  hooks  in 
the  right  respiratory  chamber,  and  the  chamber  is  then  sealed  with 
mercury.  The  left,  or  analytic  chamber,  is  now  partially  filled  with 
mercury  in  the  manner  described  elsewhere,  and  then  the  appara- 
tus is  washed  as  usual  by  air  free  from  carbon  dioxide.  The 
time  at  which  barium  hydroxide  is  introduced  into  the  top  of  the 
tube  in  the  left  chamber  is  recorded  and  the  stopcock  between 
the  two  chambers  is  closed.  When  at  the  end  of  ten  minutes  the 
drop  on  the  tube  in  the  left  chamber  is  perfectly  clear,  having  not 
a  single  granule  of  the  precipitate  visible  to  a  lens,  thus  insuring 
that  the  air  used  for  washing  is  absolutely  free  from  carbon 
dioxide,  a  known  amount  of  the  gas  from  the. right  respiratory 
chamber  is  introduced  into  the  left  chamber  in  which  the  clear  drop 
of  barium  hydroxide  has  been  exposed,  and  it  is  determined 
whether  or  not  the  amount  of  the  gas  taken  contains  enough 
carbon  dioxide  to  give  a  precipitate  in  several  minutes.  Usually 
ten  minutes  will  be  sufficient  for  the  reaction.1  If  it  does  not  give 
a  precipitate  in  this  time,  a  larger  volume  should  be  taken  until 
the  precipitate  appears  within  ten  minutes.  If  it  does,  the  ap- 
paratus is  washed,  dried,  and  with  a  fresh  nerve  the  procedure  is 
repeated,  but  a  less  volume  of  the  gas  than  the  amount  which 
before  gave  the  precipitate  is  withdrawn  into  the  left  chamber 
from  the  right.2 

In  this  way,  by  the  use  of  several  fresh  nerves,  a  minimum 
volume  of  the  gas  for  a  known  weight  of  the  nerve  which  gives  a 
precipitate  is  determined.  This  minimum  volume  should  con- 
tain a  definite  quantity  of  carbon  dioxide,  namely,  i  .oXio~~?  g., 
the  amount  carefully  determined  previously  (taking  known 
amounts  of  the  exceedingly  diluted  gas)  to  be  just  sufficient  to 
produce  a  noticeable  precipitate. 

1  The  weight  of  this  plate  is  known,  hence  the  weight  of  the  nerve 
can  be  determined  very  quickly  (see  p.  38). 
3  See  footnote,  p.  126. 


CHEMICAL  SIGNS  OF  IRRITABILITY  21 

"  Thus,  since  we  know  the  original  volume  of  the  chamber  in 
which  the  respiration  took  place  and  from  which  this  minimum 
volume  is  withdrawn,  and  since  we  know  the  quantity  of  carbon 
dioxide  contained  in  this  volume,  it  is  easily  calculated  how 
much  carbon  dioxide  is  given  off  by  the  nerve  during  the  known 
period.  It  should  be  understood  that,  in  determining  the  mini- 
mum volume  of  gas  taken  from  the  respiratory  chamber,  a  series 
of  experiments  was  conducted  in  order  to  calculate  both  the  mini- 
mum volume  which  just  gives  the  precipitate  and  the  maximum 
volume  which  does  not  give  the  precipitate  for  a  known  weight  of 
the  nerve  for  a  known  period  of  respiration.  In  Table  I,  in  the 
Appendix  (p.  128),  columns  8  and  9  refer  to  these  volumes 
calculated  from  experiments  for  10  mg.  of  the  nerve,  for  ten 
minutes. 

Medullated  nerve  fibers. — We  have  repeated  this 
experiment  with  the  sciatic  nerve  of  the  frog,  this  nerve 
being  a  typical  medullated  nerve.  The  result  showed, 
not  only  that  medullated  nerves  also  give  off  carbon 
dioxide,  but  that  they  give  a  quantity  of  about  5-5X 
io~~7  g.  for  each  10  mg.  of  the  nerve  for  the  first  ten 
minutes,  which  is  a  little  less  than  was  obtained  from 
the  non-medulla  ted  nerve. 

A  large  variety  of  nerves  was  tested  to  see  whether  or 
not  all  resting  nerves  give  off  carbon  dioxide.  As  a 
result,  we  found  no  exception  in  any  of  them,  although 
they  vary  quite  widely  in  the  rate  at  which  they 
produce  carbon  dioxide.  The  following  nerves  were 
examined,  and  it  will  be  noticed  that  the  list  includes 
all  varieties,  such  as  sensory,  motor,  vertebrate  and 
invertebrate,  medullated  and  non-medullated  nerves. 

1.  MOTOR  NERVE:    Oculomotor  nerve  of  the  skate  (Raia 
ocallatd). 

2.  SENSORY  NERVE:  Olfactory  nerve  of  the  same. 


22  A  CHEMICAL  SIGN  OF  LIFE 

3.  MEDULLATED   NERVES:  Sciatic  nerve  of  the  dog,   frog, 
turtle,  mouse,  guinea-pig;   optic  nerve  of  the  skate  (both 
Raia  ocallata  and  Raia  erinecia) . 

4.  NON-MEDULLATED  NERVES:    Nerves  of  the  spider  crab, 
olfactory  nerve  of  the  skate  (Raia  ocallata) . 

5.  NERVES  or  INVERTEBRATES:    Nerves  of  the  spider  crab, 
Limulus,  Limax. 

6.  NERVES  OF  VERTEBRATES:    Nerves  of  frog,  dog,  mouse, 
squiteague    (Cynoscion   regalis),    and    skate    (both    Raia 
ocallata  and  Raia  erinecia). 

7.  NERVES  OF  WARM-BLOODED  ANIMALS:  Those  of  dog,  rat, 
rabbit,  guinea-pig. 

8.  NERVES  OF  COLD-BLOODED  ANIMALS:  Those  of  frog,  squi- 
teague (Cynoscion  regalis),  catfish,  carp,  and  skate. 

9.  SENSORY  DENDRITE:    Lateral  line  nerve  (ramus  lateralis 
vagi)  of  carp  and  catfish,  and  ramus  lateralis  accessorius  of 
catfish. 

This  is  a  partial  list  of  the  many  nerves  examined  and 
it  is  given  only  to  show  that  we  are  justified  in  making 
the  generalization  that  all  freshly  isolated  nerves  of 
all  animals,  regardless  of  the  kind  of  nerve  or  of  the  kind 
of  animal,  produce  carbon  dioxide.  It  is  thus  certain 
that  chemical  changes  of  a  very  vigorous  kind  are 
going  on  constantly  in  this  tissue  without  any  visible 
results.  Nerves  respire;  they  are  not  chemically  inert. 
It  remains  now  for  us  to  establish  the  fact  that  this  car- 
bon dioxide  is  a  product  of  normal  metabolic  activity 
and  is  not  due  to  a  disintegration  involved  in  the  process 
of  dying  on  the  part  of  the  tissue,  or  to  a  lifeless  fermen- 
tation, and  that  it  is  not  simply  gas  which  had  happened 
to  be  absorbed  by  the  nerve  from  the  atmosphere  or  the 
blood. 

Is  this  carbon  dioxide  produced  by  living  processes  ? — 
Since  there  are  many  organic  compounds,  as  well  as  dead 


CHEMICAL  SIGNS  OF  IRRITABILITY  23 

tissues,  which  produce  carbon  dioxide  either  by  direct 
oxidation,  by  fermentation,  or  by  the  decomposition  of 
carbonates  by  acids,  the  possibility  that  this  carbon 
dioxide  which  we  have  detected  is  not  a  product  of 
vital  activity  cannot  be  so  easily  disproved.  Inasmuch 
as  our  apparatus  detects  such  a  small  amount  of  the  gas 
as  that  which  is  contained  in  one-sixth  of  a  cubic  centi- 
meter of  the  purest  air,  we  cannot  accept  the  results  just 
cited  as  certain  proof  that  the  normal  nerve  undergoes 
metabolic  changes.  We  must  inquire,  therefore,  whether 
this  carbon  dioxide  is  produced  by  living  processes.  In 
the  first  place,  as  the  biometer  in  its  present  form  cannot 
examine  the  carbon  dioxide  production  of  a  nerve  in  its 
normal  position  and  with  its  muscle  attached  to  it,  we 
have  to  use  an  isolated  nerve.  Certain  experimental 
factors  are  thus  introduced  which  must  be  carefully 
considered  before  we  interpret  our  observations.  It 
is  first  necessary  to  be  sure  that  this  isolated  nerve  lives 
and  remains  excitable  for  a  considerable  period  after  it 
has  been  removed  from  the  body.  We  can  be  quite 
certain  that  this  is  the  case  because  of  the  fortunate 
circumstance  that  the  passage  of  the  nerve  impulse 
through  such  an  isolated  nerve  produces  a  characteristic 
electrical  disturbance,  which  we  may  detect  by  a  sensi- 
tive galvanometer.  As  long  as  this  electrical  dis- 
turbance occurs  and  the  nerve  is  excited,  we  may  be 
perfectly  sure  that  the  nerve  is  living.  It  is  as  certain 
a  sign  of  the  passage  of  the  nerve  impulse,  and  conse- 
quently as  sure  an  evidence  of  the  vitality  of  the  nerve, 
as  would  be  the  contraction  of  the  muscle  which  the 
nerve  supplies,  had  this  remained  attached  to  it.  By 
thus  testing  with  a  galvanometer  isolated  nerves,  such 


A  CHEMICAL  SIGN  OF  LIFE 


as  we  have  examined,  it  has  been  found  by  Waller  that 
the  vitality  persists  even  as  long  as  nineteen  hours 
after  removal  from  the  body.  These  facts  are  proof, 
therefore,  that  the  observations  made  on  the  carbon 
dioxide  production  of  isolated  nerves  are  really  made  on 
active  living  nerves,  and  they  may  be  regarded  as  quali- 
tatively similar  to  what  would  happen  in  the  normal 
nerve  in  situ  were  we  able  to  measure  its  carbon  dioxide 
production. 

TABLE  I 

COMPARISON  BETWEEN  NORMAL  AND  KILLED  (BY  STEAM)  NERVES  OF  SPIDER  CRAB 


I 

2 

3 

4 

Cubic 

6 

7 

Date 

Tempera- 
ture of 
Room 
Degrees  C. 

Weight  of 
Nerve  in 
Milligrams 

Stimula- 
tion 

Centime- 
ters of  Gas 
Taken 
from  Res- 
piratory 

Duration 
of  Respira- 
tion 
Minutes 

Ppt.  of 
Ba(COi) 
after  Ten 
Minutes 

Chamber 

November  4  .  . 

13 

40  (killed) 

No 

o.S 

10 

_ 

November  4  .  . 

40  (killed) 

Yes 

0.5 

10 

— 

November  5  .  . 

M 

1  6  (normal) 

No 

I.O 

10 

+ 

November  6  .  . 

15 

1  6  (killed) 

No 

1.0 

12 

November  7  .  . 

16 

1  6  (normal) 

No 

I.O 

10 

+ 

If  the  carbon  dioxide  is  produced  by  vital  activity, 
its  production  should  be  diminished  when  the  nerve  is 
killed.  This  we  can  demonstrate  by  placing  a  nerve 
killed  by  steam  in  one  chamber  of  the  biometer  and  an 
equal  weight  of  a  normal  living  nerve  in  the  other 
chamber  and  then  comparing  simultaneously  the  output 
of  carbon  dioxide  in  the  living  and  dead  nerves.  It  is 
found  that  the  living  nerve  continues  to  give  off  carbon 
dioxide,  while  the  dead  gives  off  extremely  little,  the 
difference  between  the  two  becoming  more  marked  as 
time  goes  on.  Such  a  comparison  between  two  nerves 
of  the  spider  crab  is  given  in  Table  I,  from  which  it 


CHEMICAL  SIGNS  OF  IRRITABILITY  25 

will  be  seen,  if  the  experiments  on  November  6  and  7  are 
compared,  that  i  c.c.  of  gas  taken  from  the  respiratory 
chamber  in  which  the  dead  nerve  had  been  for  a  certain 
length  of  time  contained  not  enough  carbon  dioxide  to 
produce  a  precipitate,  while  i  c.c.  of  gas  from  the  cham- 
ber in  which  the  living  nerve  had  been  for  the  same  time 
did  produce  a  precipitate  and  consequently  contained 
more  carbon  dioxide.  It  is  clear,  then,  that  a  dead 
nerve  gives  off  less  carbon  dioxide  than  the  living. 

Comparison  of  anesthetized  and  normal  nerves. — By 
the  use  of  anesthetics  we  can  diminish  the  irritability, 
or,  as  we  may  say,  the  vitality,  of  the  nerve  without 
abolishing  it  altogether,  The  nerve,  although  anesthe- 
tized, is  still  alive,  but  in  a  condition  of  suspended 
animation.  When  the  anesthetic  escapes  from  it,  it 
recovers  its  normal  vitality.  If  the  carbon  dioxide  has 
been  produced  by  a  vital  process  and  is  at  all  corre- 
lated with  the  state  of  irritability  of  the  nerve,  we 
should  expect  that  a  diminution  of  that  irritability  by 
anesthetics  would  produce  a  diminution  in  the  carbon 
dioxide  output.  If,  on  the  other  hand,  this  carbon 
dioxide  is  the  result,  not  of  a  vital  process,  but  of  a 
fermentation,  or  of  an  acid  production  of  some  sort,  then 
we  should  expect  that  it  would  be  little,  if  at  all,  affected 
by  the  anesthetic.  Accordingly,  nerves  were  anesthe- 
tized in  various  ways,  for  example,  by  placing  them  in  a 
solution  of  urethane,  or  they  were  treated  with  the 
vapors  of  ether,  or  the  nerve  was  isolated  from  a  deeply 
anesthetized  frog,  and  the  quantity  of  carbon  dioxide 
produced  by  such  nerves  was  compared  with  the  quantity 
produced  by  normal  nerves  of  the  same  animals.  It  was 
found  always  that  the  anesthetized  nerve  gave  off 


26  A  CHEMICAL  SIGN  OF  LIFE 

decidedly  less  carbon  dioxide  than  the  nerves  of  normal 
frogs  or  nerves  taken  from  frogs  whose  circulation  had 
been  suspended  for  a  period  of  time  equal  to  that  of 
etherization.  A  perfect  parallelism  was  found  to  exist 
between  the  carbon  dioxide  production  and  the  state 
of  excitability  of  the  nerve.  Thus  small  quantities  of 
anesthetics  have  often  the  effect  of  increasing  at  first 
the  excitability  of  the  nerve,  and  it  was  found  that  such 
quantities  also  produced  at  first  an  increase  in  the  carbon 
dioxide.  A  further  consideration  of  the  effects  of 
anesthetics  on  the  metabolism  of  the  claw  nerve  of  the 
spider  crab  will  be  found  in  chapter  iv.  The  important 
fact  is  that  since  these  agents  are  known  to  affect  the 
normal  uncut  nerve  in  situ  and  also  to  modify  carbon 
dioxide  production  in  an  isolated  nerve,  and  in  a  manner 
parallel  with  their  known  actions  on  irritability,  it  is 
certain  that  at  least  the  larger  part  of  the  carbon  dioxide 
we  measure  in  an  isolated  resting  nerve  must  have  been 
produced  by  a  physiological  process. 

Carbon  dioxide  production  in  a  hydrogen  atmosphere .— 
Although  many  nerves  remain  alive  for  a  long  time  in  an 
atmosphere  free  from  oxygen,  they  generally  exhibit  a 
lowered  irritability  when  compared  with  nerves  in  normal 
air.  It  has  been  found,  for  example,  that  if  nerves 
remain  in  the  body  after  the  circulation  of  a  frog  has 
ceased,  so  that  they  have  not  been  supplied  with  oxygen 
for  some  time,  they  are  by  no  means  so  easily  stimulated 
by  a  salt  solution  as  are  normal  nerves.  Their  vitality 
is  reduced.  A  similar  change  occurs  in  nerves  taken 
out  of  the  body  and  put  in  hydrogen  gas.  In  them,  also, 
irritability  is  decidedly  diminished.  If,  now,  carbon 
dioxide  is  produced  in  these  nerves  by  a  vital  process, 


CHEMICAL  SIGNS  OF  IRRITABILITY  27 

we  should  expect  to  find  that  less  carbon  dioxide  was 
produced  by  nerves  in  an  atmosphere  of  hydrogen  than 
in  normal  air.  On  the  other  hand,  if  the  carbon  dioxide 
was  due  to  some  fermentation,  or  non- vital  process,  then 
it  should  not  be  influenced  by  the  absence  of  oxygen. 

When,  with  Dr.  Adams,  we  determined  the  rate  of 
carbon  dioxide  production  in  nerves  placed  in  an  atmos- 
phere of  hydrogen  gas,  care  having  been  taken  to  insure 
the  gas  being  perfectly  pure,  we  found  that  the  rate  was 
only  about  half  that  of  the  normal  nerve.  It  appears 
from  this  determination  that  in  a  medium  deficient  in 
oxygen  the  claw  nerve  of  the  spider  crab  gives  off  less 
carbon  dioxide  than  in  an  ordinary  atmosphere.  The 
effect  cannot  be  due  to  the  hydrogen,  since  that  gas  has 
no  physiological  action,  but  is  quite  inert,  and  we  may 
conclude  that  the  lowering  of  the  carbon  dioxide  is  due 
to  the  lack  or  absence  of  oxygen.  This  is  additional 
evidence  that  the  lowering  of  the  gaseous  output  is  a 
physiological  phenomenon,  and  that  the  carbon  dioxide 
measured  in  normal  isolated  nerves  is  a  product  of  normal 
metabolism,  and  is  not  the  mere  diffusion  outward  of  the 
gas  which  is  present  in  the  tissue,  being  produced  there 
by  other  than  living  processes. 

Carbon  dioxide  production  of  the  isolated  nerve  at  suc- 
cessive time  intervals. — If  the  carbon  dioxide  production 
is  due  to  a  vital  process,  it  might  be  expected  to  diminish 
gradually  in  the  isolated  nerve  as  its  vitality  diminishes. 
On  the  other  hand,  there  was  a  possibility  that  the  iso- 
lated nerve  had  become  infected  with  bacteria  and  that 
the  carbon  dioxide  might  be  due  to  their  action.  If 
this  were  the  case,  it  would  be  expected  that  the  carbon 
dioxide  would  gradually  increase.  Accordingly,  experi- 


28 


A  CHEMICAL  SIGN  OF  LIFE 


ments  were  tried  to  discover  how  the  carbon  dioxide 
production  behaved  at  successive  time  intervals  after 
the  nerve  was  removed  from  the  body.  A  number  of 
sciatic  nerves  were  isolated  from  several  frogs  of  the  same 
size  and  sex  and  were  left  for  varying  periods  of  time  in 
Ringer's  salt  solution,  in  which  they  live  well.  The 
rate  of  the  gas  production  was  then  determined  in  the 
nerves  when  removed  from  the  Ringer  solution  after 
one  hour,  two  hours,  and  at  other  intervals  up  to 
twenty-five  hours.  The  interesting  results  given  in 
Table  II  make  it  clear  that  the  fresh  nerve  produces  the 

TABLE  II 
SHOWING  DECREASED  COj  PRODUCTION  BY  LONG  STANDING  (FROG'S  SCIATIC) 


I 

2 

3 

4 

Minimum  Cubic 

Temperature 
of  Room 
Degrees  C. 

Time  Elapsed 
after  Isolation 

Centimeters  Neces- 
sary to  Give  X 
Calculated  for 
10  mg.  for  10 

Total  Amount  of 
COi  Produced  by 
10  mg.  of  Nerve  in 
10  Minutes 

Minutes 

24 

Immediately 

27    c  c 

5  5X10    7  g  (XL 

25  

i      hour 

7.08  c.c. 

2.iXio~7  g.  COi 

24 

2      hours 

10  8    c.c. 

I    4XlO~7  g    (XL 

24  

5.5  hours 

12.8     C.C. 

i.iXio~7  g.  COi 

23.5  

7      hours 

IS-3      C.C. 

o.gXio~7  g.  CQi 

23  5 

10  5  hours 

21    O     C  C 

o  6Xio~7  g  (XL 

24  

26      hours 

9        c.c.  * 

i.6Xio~7  g.  (XL 

24 

27  4  hours 

i  8    c.c. 

8  iXio~7  g  (XL 

*  The  gradual  increase  at  this  point  should  be  noted  (after  26  hours,  it  is  clear  that 
bacterial  decomposition  sets  in). 

most  carbon  dioxide  and  that  the  amount  produced  per 
unit  of  time  interval  decreases  rapidly  up  to  about 
twenty-three  hours  and  from  then  on  suffers  a  very 
rapid  increase.  These  facts  show  that  the  carbon  dioxide 
output  diminishes  as  the  vitality  of  the  nerve  diminishes, 
and  that  as  bacterial  decomposition  sets  in  there  is  a 
sudden  and  rapid  increase.  There  is,  therefore,  a 


CHEMICAL  SIGNS  OF  IRRITABILITY  29 

parallelism  between  the  decrease  in  metabolism  and 
decrease  of  irritability  in  the  nerve.  The  gas  produc- 
tion slows  up  as  the  nerve  approaches  death.  This 
indicates,  also,  that  the  carbon  dioxide  is  formed  by  a 
vital  process. 

Comparison  between  the  metabolism  of  resting  nerves 
and  other  tissues. — While  a  comparison  of  the  rate  of  the 
metabolism  of  the  nerve  with  that  of  other  tissues  is 
subject  to  a  good  many  limitations,  since  there  are  so 
many  and  great  variations  in  conditions  which  do  not 
affect  all  tissues  similarly,  it  is  nevertheless  interesting 
to  note  whether  the  nerve  respires  relatively  more  or  less 
than  most  other  tissues.  In  order  to  give  a  better 
numerical  picture  of  the  amount  of  metabolism  in  the 
resting  nerve,  as  compared  with  other  tissues,  we  have 
set  down  in  Table  III  the  figures  for  carbon  dioxide 
production  in  various  animals.  Since  there  are  no  exact 
determinations  made  of  the  carbon  dioxide  production  of 
the  spider  crab  as  a  whole,  or  of  its  tissues,  we  have  used 
for  comparison  various  other  Crustacea  where  these 
data  have  been  determined.  It  will  be  noticed  from  an 
inspection  of  this  table  that  the  spider  crab  nerve  pro- 
duces, weight  for  weight,  carbon  dioxide  at  a  rate  three 
to  four  times  that  of  the  whole  body  of  crabs,  and  almost 
as  much  in  proportion  to  weight  as  a  human  being  at 
rest.  Recently  Bayliss,  in  his  admirable  book  entitled 
Principles  of  General  Physiology,  expressed  a  doubt  of  our 
figures.  He  thinks  that  the  gas  we  measured  must  be 
due  to  some  cause  other  than  the  metabolic  activity  of 
the  nerve,  because,  he  says,  the  data  show  that  it  is 
greater  than  that  of  an  equal  weight  of  muscle.  It  is 
rather  difficult  for  us  to  understand  the  force  of  this 


A  CHEMICAL  SIGN  OF  LIFE 


criticism,  since  there  are  many  other  evidences  that  the 
metabolism  of  the  nervous  system  is  more  intense  than 
that  of  any  other  tissue  of  the  body.  There  is,  as  far 
as  we  know,  no  physiological  reason  for  assuming  a 
priori  that  the  nerve  has  a  lower  metabolism  than  other 
tissues,  but,  on  the  contrary,  the  direct  and  indirect 

TABLE  III 


Animals 

CO,  per 
Kilogram 
per  Hour 

Temperature 
Degrees  C. 

Determined  by  * 

Crustacea  (whole  animal)  
Crayfish  (Astacus) 

Jolyet  and  Regnaut 

Crab  (Cancer  pagurus)  
Lobster  (Homarus  vulgaris)  
Nerve  of  spider  crab  (Labinia  cana- 

89.  9  c.c. 
54-  4  c.c. 

16 
15 

:    :     : 

liculata)  

212  c.c.  or 

15-16 

Tashiro 

0.402  g. 

Frog: 

(Rana  esculenta)  (whole  animal).. 

o.o82g. 

i? 

Schultz 

(Rana  temporaria)  (whole  animal) 
(Rana  pipiens)  (sciatic  nerve)  .... 

o.355g- 
o.33    g- 

19-20 
15 

Pott 
Tashiro 

(Rana      temporaria^)       (isolated 

muscle)  

0.18    g. 

21 

Fletcher 

Dog  

i  325  g 

Regnaut  and  Reiset 

Man  at  rest  

0.41    g. 

Pettenkoffer  and  Voit 

O.OI     g. 

a      u       u 

0.37  g- 

Speck 

*  All  the  figures  are  quoted  from  Schafer's  Text  Book  of  Physiology,  I,  702,  707,  and 
708,  except  that  of  the  isolated  muscle,  which  I  calculated  from  Fletcher  (op.  «'/.). 
Fletcher  fails  to  state  the  weight  of  a  leg,  but  gives  the  value  0.2  c.c.  for  one-half  hour. 
Hill  believes  that  if  we  take  each  leg  as  6  g.  in  average,  the  value  will  not  be  far  from  the 
truth. 

t  Fletcher  fails  to  state  the  species  of  the  frog,  but  it  is  inferred  from  Hill's  paper. 

evidence  shows  that  it  has  a  more  intense  metabolism. 
It  is  no  doubt  true,  however,  that  an  isolated  nerve,  such 
as  we  have  used,  respires  somewhat  faster  than  the 
same  nerve  intact  in  the  body,  because  the  effect  of 
cutting  the  nerve  is  to  act  as  a  stimulant.  But,  even 
allowing  for  this  effect,  the  metabolism  still  remains 
markedly  higher  than  that  of  most  other  tissues.  We 
may  add  here,  however,  that  the  hourly  rate  of  output 
of  carbon  dioxide  from  the  resting  nerve  of  a  frog  is 


CHEMICAL  SIGNS  OF  IRRITABILITY  31 

at  a  maximum  only  about  o .  03  per  cent  of  the  wet  weight 
of  the  tissue. 

Comparison  of  carbon  dioxide  output  of  nerve  fibers  and 
nerve  ganglia. — From  the  table  already  presented  it  is 
clear  that  the  living  nerve  trunk  containing  no  nerve 
cells  gives  off  carbon  dioxide  at  a  rapid  rate.  It  is  inter- 
esting to  see  whether  nerve  tissues  containing  ganglion 
cells  produce  more  or  less  carbon  dioxide  per  gram  per 
hour  than  the  nerve  fibers.  For  this  purpose  we  studied 
the  ganglionated  nerve  cord  on  the  back  of  the  heart 
of  the  king  crab  (Limulus  polyphemus).  This  is  an 
elongated  automatic  ganglion  which  has  been  shown 
to  be  the  direct  cause  of  the  heart-beat.  It  was  isolated 
carefully  from  the  heart,  the  operation  taking  but  a  few 
minutes,  placed  in  the  biometer,  and  its  carbon  dioxide 
output  measured.  It  was  found  to  give  2.3Xio~7  to 
4 . 7  X  io~7  g.  CO2  per  centigram  per  ten  minutes  at  22.8° 
to  23°  C.  The  rate  was  somewhat  lower  in  the  larger 
individuals,  which  were  usually  females.  This  amount 
of  carbon  dioxide  is  very  small  when  compared  with  the 
output  of  the  claw  nerve  of  the  spider  crab,  which  with- 
out stimulation  gives  off  from  an  equal  weight  of  tissue 
6.7Xio~7  g.  If,  however,  the  comparison  be  made 
with  the  claw  nerve  or  with  the  optic  nerve  of  Limulus 
itself — the  same  animal  as  that  from  which  the  ganglion 
was  taken — the  rate  in  the  ganglion  is  found  to  be  about 
the  same  as  that  in  the  fibers.  The  claw  nerve  of 
Limulus  gives  only  about  2 .  6X  io~7  g.  of  carbon  dioxide, 
while  the  optic  nerve  gives  somewhat  more,  namely,  2 . 6  to 
5X  io~7  g.,  depending  on  what  portion  of  the  optic  nerve 
is  taken  (see  p.  76).  Limulus  is  a  very  sluggish,  slow- 
moving  animal,  whereas  the  spider  crab  is  more  active. 


32 


A  CHEMICAL  SIGN  OF  LIFE 


It  appears  from  these  determinations  that  the  heart 
ganglion  gives  off  about  the  same  amount  of  carbon 
dioxide  per  gram  of  its  substance  as  the  nerve  fibers  of 
the  same  animal.  Certainly  there  is  no  marked  superi- 
ority of  carbon  dioxide  output  by  the  ganglion.  If 
anything,  its  rate  is  a  little  lower.  This  is  very  in- 
teresting because,  as  already  stated,  this  ganglion  is 

TABLE  IV 

SUMMARY  OF  CARBON  DIOXIDE  PRODUCTION  FROM  VARIOUS  NERVE  TISSUES 


Amount  of 

Animal 

Sex 

Nerve 

Temper- 
ature 
Degrees  C. 

COa  Given 
Off  by  10  mg. 
of  Nerve  in 

Estimated 
By 

10  Minutes 

$ 

Nerve  cord  of  heart  (30- 

34  mg.)  

23-23  •  5 

4.7X10—  7  g. 

Tashiro, 

$ 

Nerve  cord  of  heart   (51 

Adams 

mg.)  

23 

2.4X10"  7g. 

Tashiro, 

$ 

Nerve  cord  of  heart  (52 

Adams 

Limulus 

mg.) 

23 

2.3X10"  7g. 

Tashiro, 

Polyphemus 

$ 

Claw  nerve  

23 

2.  6Xio-7  g. 

Adams 
Tashiro, 

Adams 

? 
? 

Optic  nerve,  whole  
Optic  nerve,  proximal  part 

17.8 
22.5 

2.6Xio-7g. 
3.0X10     7g. 

Tashiro 
Tashiro 

2 

Optic  nerve,  distal  part  .  . 

22 

5.0X10     7g. 

Tashiro 

Claw  nerve,  whole 

I5-I6 

6.7Xio~7g. 

Tashiro 

Claw  nerve,  whole  

20.2 

7.9Xio~-7g. 

Tashiro, 

Adams 

Labinia 

Claw  nerve,  proximal  part 

21.4 

8.oXio"~7g. 

Tashiro 

canaliculata 

Claw  nerve,  distal  part  .  .  . 

23.2 

3.7Xio"7g. 

Tashiro 

Claw  nerve,  whole,  when 

stimulated  

I4-l6 

16.0X10—  7g. 

Tashiro 

( 

Sciatic  resting 

19—  20 

5.5X10—  7g. 

Tashiro 

Rana  Piptens     < 

Sciatic,  stimulated  

20-22 

i4.2Xio"7g. 

Tashiro 

automatically  active  all  the  time  and  is  constantly  dis- 
charging nerve  impulses.  Of  course  this  result  may 
be  due  either  to  an  equality  of  the  metabolism  in  cells 
and  fibers,  or  the  injury  may  have  raised  the  rate  more 
in  the  nerve  than  in  the  ganglion,  or  in  the  ganglion  the 
amount  of  non-nervous  tissue  may  be  somewhat  greater 
than  in  the  nerve  trunk,  so  that  the  carbon  dioxide  pro- 


CHEMICAL  SIGNS  OF  IRRITABILITY  33 

duction  per  gram  is  reduced  thereby.  But  one  thing 
seems  to  be  certain,  i.e.,  that  to  attribute  the  carbon 
dioxide  production  in  the  nerve  fiber  to  the  connective 
tissue  cells  surrounding  the  nerve  trunk,  as  Bayliss  does, 
is  rather  ridiculous.  Nerve  cells  evidently  breathe 
at  about  the  same  rate  as  nerve  fibers,  and  not  faster,  as 
one  might  suppose.  Table  IV  summarizes  the  carbon 
dioxide  production  by  various  nervous  tissues,  some  of 
which  contain  cells  and  others  only  fibers. 

Summary. — We  have  thus  far  shown,  then,  that  the 
living  nerve  fiber  is  no  exception  to  the  rule  that  all 
living  matter  undergoes  chemical  changes.  It  respires. 
There  is  a  chemical  sign  of  irritability  in  the  nerve. 
By  the  use  of  a  proper  apparatus  of  sufficient  delicacy 
we  can  demonstrate  experimentally  the  formation  of 
carbon  dioxide  in  all  nerves;  and  by  estimating  the 
amount  produced  under  various  conditions — conditions 
which  we  know  affect  the  state  of  irritability  of  the 
normal  nerve  in  the  body — we  find  a  very  close  parallel- 
ism between  the  amount  of  carbon  dioxide  produced  and 
the  state  of  irritability.  We  are  justified,  therefore,  in 
concluding  that  the  gas  thus  measured  is  the  expression 
of  the  metabolic  activity  of  the  nerve.  We  may  now 
pass  on  to  discover  whether  this  carbon  dioxide  is 
increased  in  case  of  stimulation. 


CHAPTER  III 

CHEMICAL  SIGNS  OF  IRRITABILITY  IN  THE  NERVE 
FIBER—  Continued 

Increased  metabolism  on  stimulation. — We  have 
already  stated  that  all  living  matter,  whether  it  is  an 
organism  or  an  isolated  tissue,  normally  undergoes 
chemical  changes  and  produces  carbon  dioxide  as  one 
of  the  final  products  of  its  metabolic  activity,  and  that 
the  nerve  fiber  is  no  exception  to  this  rule.  In  other 
words,  respiration  is  one  of  the  unfailing  signs  of  life 
and  is  a  necessary  condition  for  living  processes.  But 
carbon  dioxide  production  from  a  tissue  is  not  by  itself 
a  sufficient  sign  of  life.  For  there  are  many  chemical 
compounds  which  spontaneously  give  off  carbon  dioxide, 
among  others  sea-water,  bicarbonate  solutions,  as  well 
as  organic  materials  which  are  unstable.  It  would 
obviously  be  a  mistake  to  call  these  compounds  living 
because  of  the  fact  that  they  give  off  this  gas.  This 
criterion  alone,  therefore,  cannot  be  used  for  detecting 
the  vitality  of  the  tissues. 

Not  only  is  it  common  for  many  non-living  matters  to 
give  off  carbon  dioxide  spontaneously,  but  there  are 
also  some  whose  mode  of  gaseous  exchange  is  remarkably 
similar  to  that  of  the  living  process.  Among  these 
substances  there  is  none  in  which  the  parallelism  to 
vital  respiration  is  more  detailed  and  interesting  than 
ordinary  linseed  oil.  The  many  curious  resemblances 
of  the  chemical  processes  involved  in  painting  to  proto- 

34 


CHEMICAL  SIGNS  OF  IRRITABILITY  35 

plasmic  respiration  and  growth  have  already  been 
pointed  out.1  It  is  unnecessary  to  go  into  this  more 
fully  than  to  call  attention  to  the  facts  that  linseed  oil 
takes  up  oxygen,  that  it  gives  off  carbon  dioxide,  that  it 
is  stimulated  by  light,  that  it  undergoes  also  other 
phases  of  metabolism  common  to  living  matter,  and 
that,  very  singularly,  it  exhibits  many  of  the  phenomena 
of  memory,  learning,  and  forgetting.  It  is  striking, 
too,  that  this  respiration  is  of  an  autocatalytic  nature— 
that  is,  it  becomes  more  rapid  as  it  progresses  and  in  this 
respect  resembles  the  psychic  phenomena  of  memory  and 
learning.  Thus  we  see  that  respiration  alone  cannot 
be  taken  as  a  criterion  of  life;  and,  furthermore,  that 
even  the  characteristic  features  of  protoplasmic  respira- 
tion itself  cannot  be  said  to  be  peculiar  to  living  things. 
A  more  certain  criterion  of  life  is  the  increase  of  respira- 
tion on  stimulation. 

It  is  well  known  that  contracting  muscle  produces 
more  carbon  dioxide  than  resting  muscle.  We  breathe 
faster  when  we  run.  We  can  measure  the  irritability  of 
the  muscle  by  its  increased  metabolism  occurring  on 
stimulation.  Is  it  possible  to  increase  the  metabolism 
of  the  nerve  also  by  stimulation  ?  Can  the  nerve,  one 
of  the  most  irritable  tissues  of  all,  perform  its  function 
without  consuming  any  material  ?  Is  the  nerve  impulse 
something  similar  to  an  electrical  current  passing 
through  a  rather  imperfect  conductor  ?  How  is  the 
electromotive  force  created  in  the  nerve  fiber  when  the 
impulse  passes  through  it?  Is  it  simply  the  equiva- 
lent of  the  energy  we  put  in  at  the  initial  stimulation  ? 
These  questions  cannot  be  considered  unless  we  first 

1  Mathews,  Textbook  of  Physiological  Chemistry,  1915,  p.  67. 


36  A  CHEMICAL  SIGN  OF  LIFE 

determine  positively  whether  or  not  nervous  functions 
involve  metabolic  change. 

As  far  as  the  brain — the  master  nervous  tissue  of  the 
body — is  concerned,  it  is  perfectly  obvious  that  its  action 
involves  a  very  intense  chemical  activity.  This  is 
shown  by  various  circumstances.  It  is  made  evident 
in  the  first  place  by  the  fact  that  the  brain  has  an 
extremely  abundant  blood  supply  and  that  the  blood 
returning  from  the  brain  has  lost  a  considerable  part 
of  its  oxygen.  Direct  measurement  of  the  amount  of 
oxygen  actually  consumed  by  the  brain  shows  that  it  is 
greater  than  that  of  any  other  tissue  in  the  body  relative 
to  its  weight.  The  carbon  dioxide  production  is  also 
greater.  Everyone  knows,  also,  that  keen  intellectual 
work  depends  on  a  plentiful  blood  supply  to  the  head. 
When  one  works  hard  intellectually  the  face  flushes; 
often  the  hands  and  feet  become  cold,  owing  to  con- 
centration of  the  blood  in  the  head.  If  this  increase  of 
blood  does  not  occur,  keen  intellectual  effort  is  impos- 
sible. If  the  circulation  stops,  or  even  if  the  blood 
pressure  becomes  low,  we  become  unconscious  or  faint. 
These  facts  are  sufficient  to  prove  that  the  functions  of 
the  brain,  at  any  rate,  involve  oxygen  and  are  expressed 
in  its  respiration.  The  attempt  to  measure  the  amount 
of  heat  produced  in  the  brain  during  intellectual  effort 
has  thus  far  been  unsuccessful,  owing  in  part  to  the  fact 
that  it  is  impossible  ever  to  get  the  brain  in  a  state  of 
rest.  It  is  always  in  partial  activity.  In  the  second 
place,  the  brain  makes  only  a  small  portion  of  the  total 
weight  of  the  body,  so  that  its  heat  makes  but  a  small 
fraction  of  that  of  the  whole  body,  and  it  is  this  which 
we  have  to  measure.  It  has  been  observed,  also,  that 


CHEMICAL  SIGNS  OF  IRRITABILITY  37 

if  Ehrlich's  method  of  staining  tissues  with  methylene 
blue  is  used,  a  spot  of  the  surface  of  the  brain  loses  its 
blue  color  when  it  is  stimulated,  owing  to  the  consump- 
tion of  oxygen  and  the  resulting  decolorization  of  the 
blue. 

We  may  now  consider  the  carbon  dioxide  output  of 
nerves  on  stimulation. 

N on-medullated  nerves. — The  biometer  is  so  delicate 
that  in  trying  these  experiments  many  precautions  had 
to  be  taken  to  make  certain  that  the  experimental  con- 
ditions themselves  did  not  produce  an  increase  of 
carbon  dioxide  independent  of  the  change  in  the  metabo- 
lism of  the  nerve.  If  we  stimulate  with  an  electrical 
current,  we  have  to  be  on  our  guard  lest  there  should  be 
direct  decomposition  of  some  substances  at  the  elec- 
trodes, resulting  in  more  production  of  carbon  dioxide. 
But  by  trying  various  kinds  of  stimulation,  mechanical 
and  chemical  as  well  as  electrical,  we  can  throw  out 
these  possible  sources  of  error.  We  found,  in  the  first 
place,  that  there  was  no  appreciable  increase  of  carbon 
dioxide  due  to  any  direct  electrical  decomposition  by 
stimulating  a  dead  nerve.  In  all  the  quantitative 
experiments  which  follow,  the  current  for  stimulating 
was  so  small  as  to  be  barely  perceptible  to  the  tongue. 
The  heating  effect  was,  therefore,  practically  negligible. 

A  nerve  of  the  claw  of  the  spider  crab  was  isolated 
as  before.  A  comparative  estimate  was  first  made. 
Two  pieces  of  the  nerve  of  equal  weights  and  lengths 
were  placed  separately  on  the  glass  plates,  each  nerve 
being  laid  across  the  electrodes  of  the  plate,  in  the  man- 
ner shown  in  Fig.  2.  In  this  way  either  nerve  can  be 
stimulated  at  will.  These  glass  plates  are  hung  upon  the 


A  CHEMICAL  SIGN  OF  LIFE 


platinum  wires  fused  into  the  side  of  the  apparatus  (see 
p.  113),  these  wires  being  connected  in  turn  with  the 
induction  coil.  Under  this  condition,  when  neither 
nerve  is  stimulated,  the  amount  of  the  precipitate  is. 
equal  in  the  two  chambers.  However,  when  one  of  the 
nerves  is  electrically  stimulated  with  a  weak  induction 
current,  the  distance  between  the  primary  and  second- 
ary coils  being  more  than 
10  cm.,  an  ordinary  dry 
battery  being  used,  not 
only  does  the  precipitate 
appear  sooner  in  the  cham- 
ber containing  the  stimu- 
lated nerve,  but  the 


FIG.  2. — Glass  weighing  plate. 
A,  B,  platinum  wires  fused  in  the 
rear  of  the  glass  plate,  with  hooks; 
C,  the  nerve  which  is  stimulated 
at  D;  G,  the  plate  proper.  Another 
piece  of  glass,  exactly  counter- 
balanced with  this  plate,  is  used, 
so  that  any  wet  tissue  can  be 
weighed  very  quickly. 


quantity  of  the  carbonate 
is  much  greater.  This 
difference  in  carbon  diox- 
ide production  can  be 
brought  out  better  in  the 


quantitative  estimate  made 
in  the  manner  described  above. 

As   shown   in   Table   V,   a   stimulated   nerve   fiber 
of  the  spider  crab  gave  i6Xio~~7  g.  of  carbon  dioxide  for 


TABLE  v 


Nerve 

Amount  of  CO*  Pro- 
duced by  10  mg.  of 
Resting  Nerve  in 
10  Minutes 

Amount  of  CO*  Pro- 
duced by  10  mg.  of 
Stimulated  Nerve  in 
10  Minutes 

Rate  of 
Increase 
of  CO, 

Non-medullated    (spider 
crab)  
Medullated  (frog)  

6.7Xio-7g.  (i5°-i60) 
5.5Xio-7  g.  (i9°-2o0) 

16.    Xio-7  g.  (i4°-i6°) 
14.2X10—7  g.  (2o°-22°) 

2  .  4  times 
2  .  6  times 

10  mg.   of  the  nerve  for  ten  minutes,   while  a  fresh 
unstimulated    nerve    of   the   same    animal   gave   only 


CHEMICAL  SIGNS  OF  IRRITABILITY  39 

6l7Xio~7g.  for  the  same  units.  In  other  words,  the 
output  was  increased  between  100  and  200  per  cent  by 
the  tetanization  of  the  nerve. 

Electrical  stimulation  of  medullated  nerves. — The  fact 
that  the  increased  production  of  carbon  dioxide  on 
stimulation  is  not  limited  to  the  non-medullated  nerve 
is  shown  by  our  quantitative  determination  on  the 
sciatic  nerve  of  the  frog.  Ten  milligrams  of  frog's  nerve 
gave  i4.2Xio~7  g.  of  the  gas  during  ten  minutes  of 
stimulation  as  compared  with  5.5Xio~7  g.,  the  amount 
produced  by  the  resting  nerve  of  the  same  animal.  Here 
again  stimulation  increased  the  output  from  200  to  300 
per  cent. 

Other  stimulation. — We  have  now  established  the 
fact  that  when  a  nerve  is  stimulated  by  an  electrical 
current  it  gives  off  more  carbon  dioxide.  In  order  to 
test  whether  this  increased  production  of  the  gas  on 
electrical  stimulation  is  due  to  the  direct  decomposing 
influence  of  the  current  or  to  the  state  of  excitation 
produced  by  the  stimulus,  many  additional  facts  must 
be  sought.  In  the  first  place,  if  the  increased  gas  pro- 
duction is  not  due  to  a  change  in  rate  of  metabolism, 
but  to  the  current  itself,  then  we  should  expect  that 
the  stimulation  of  a  killed  nerve  ought  also  to  cause  more 
gas  production,  provided,  of  course,  that  we  may  assume 
that  the  conditions  under  which  electrical  decomposition 
takes  place  are  the  same  in  the  living  and  in  the  dead. 

When  we  place  two  nerves  killed  by  steam,  one  in  each 
chamber  of  the  biometer,  and  stimulate  one  of  them,  the 
stimulated  nerve  does  not  give  off  more  carbon  dioxide 
than  the  unstimulated  when  the  same  strength  of  current 
is  employed  as  was  used  in  the  other  experiments. 


40  A  CHEMICAL  SIGN  OF  LIFE 

In  the  next  place,  it  was  -thought  possible  that  our 
assumption  that  the  condition  under  which  an  electrical 
decomposition  takes  place  is  the  same  in  a  living  and 
dead  nerve  may  not  be  strictly  true,  but  that  an  electrical 
current  can  in  some  way  drive  away  carbon  dioxide  more 
quickly  in  the  living  nerve.  Since  killing  by  steam  may 
also  drive  out  the  gas  already  present  in  the  tissue,  the 
apparent  indifference  of  the  dead  nerve  toward  electrical 
stimulation  may  not  prove  that  the  increased  carbon 
dioxide  accompanying  stimulation  in  the  living  nerve  is 
really  a  direct  result  of  a  change  in  its  vitality,  rather 
than  an  indirect  result  of  the  passage  of  the  electrical  cur- 
rent. If,  however,  this  increased  gas  production  were 
due  to  the  mere  electrical  decomposition,  which  was  not 
limited  to  the  point  of  contact  with  the  electrode,  we 
ought  in  the  living  nerve  to  get  a  proportional  increase  of 
carbon  dioxide  by  increasing  the  length  of  nerve  through 
which  the  current  directly  passes.  The  fact  is,  however, 
that  we  can  produce  an  increase  of  carbon  dioxide  by 
stimulating  with  electrodes  2  mm.  apart,  so  that  only  a 
small  portion  of  the  nerve  is  traversed  by  the  current, 
as  well  as  by  electrodes  15  mm.  apart.  It  makes  no 
difference  how  much  of  the  nerve  is  traversed  by  the 
current.  But  it  does  make  a  difference  how  much  of 
the  nerve  is  traversed  by  the  nerve  impulse.  These 
experiments  suggest  very  strongly  that  the  increase  of 
carbon  dioxide  on  electrical  stimulation  is  due  to  the 
increased  metabolic  activity  during  functional  activity 
in  the  nerve,  and  is  not  due  to  the  influence  of  the 
electrical  current  as  such.  With  the  aid  of  other 
means  of  stimulation  we  shall  now  proceed  to  prove 
that  all  stimulation  is  accompanied  by  an  increase  of 


CHEMICAL  SIGNS  OF  IRRITABILITY  41 

metabolic  activity,  as  shown  by  the  output  of  carbon 
dioxide. 

Mechanical  stimulation. — Since  the  ordinary  method 
for  mechanical  stimulation  cannot  be  used  directly  on 
the  nerve  in  the  biometer  in  its  present  form,  in  view  of 
the  fact  that  the  chamber  has  to  be  kept  shut,  we  used 
a  different  method,  namely,  that  of  injuring  the  nerve 
by  crushing.  That  when  protoplasm  is  smashed  vigor- 
ous chemical  changes  result  is  well  established.  Fletcher 
reports  that  injured  muscle  gives  off  more  carbon  dioxide 
than  normal  muscle;  later  he  and  Hopkins  discovered 
that  muscle  under  a  similar  condition  is  richer  in  lactic 
acid.  Mathews  observed  a  similar  increase  in  carbon 
dioxide  in  the  crushed  eggs  of  Arbacia.  We  have 
discovered  that  if  a  nerve  is  crushed  with  a  rough  edge 
of  a  glass  rod  it  gives  off  more  carbon  dioxide  than  the 
normal  one;  that  is,  an  injury  increases  the  carbon 
dioxide  output  of  the  nerve.  Since  this  increase  of 
carbon  dioxide  cannot  be  produced  by  crushing  an 
unexcitable  nerve,  we  consider  this  injury  to  be  a  form 
of  mechanical  stimulation.  (For  further  consideration 
of  this  subject  see  p.  91.) 

Chemical  stimulation. — The  study  of  the  nature  of 
chemical  stimulation  has  been  so  thoroughly  made  that 
it  might  seem  ideal  to  study  quantitatively  the  increased 
production  of  the  gas  following  the  stimulation  of  the 
nerve  by  various  salt  solutions.  But  there  are  com- 
plications which  seriously  interfere  with  the  use  of  this 
method.  We  found,  for  instance,  that  the  presence  of 
minute  quantities  of  a  foreign  liquid  is  a  seriously 
disturbing  factor  for  a  quantitative  estimate  of  carbon 
dioxide.  Qualitatively,  however,  we  found  various 


42  A  CHEMICAL  SIGN  OF  LIFE 

evidences  which  establish  the  fact  that  the  nerve  chemi- 
cally stimulated  gives  off  more  carbon  dioxide,  and  that 
when  rendered  less  excitable  by  reagents  it  produces 
less  carbon  dioxide  than  the  normal  resting  nerve. 

When  the  two  sciatic  nerves  of  a  frog  are  isolated  and 
one  is  left  in  physiological  salt  solution — 0.75  per  cent 
sodium  chloride — and  the  other  in  the  body  of  the  frog 
for  the  same  length  of  time,  and  when  they  are  trans- 
ferred to  the  two  chambers  of  the  apparatus,  it  is  found, 
if  the  quantities  of  the  carbonate  precipitates  are  com- 
pared, that  the  nerve  which  has  been  in  the  saline  solu- 
tion produces  more  carbon  dioxide  than  that  which  has 
remained  in  the  body.  It  is  known  that  such  a  saline 
solution  raises  irritability  and  ultimately  stimulates  the 
frog's  sciatic  nerve. 

The  different  rates  at  which  carbon  dioxide  is  pro- 
duced from  different  nerves  treated  by  various  con- 
centrations of  potassium  chloride  are  equally  instructive. 
When  a  nerve  is  placed  in  a  molecular  solution  of 
potassium  chloride,  stimulation  takes  place  for  a  con- 
siderable time.  Then  finally  the  nerve  becomes  inex- 
citable.  But  if  the  nerve  is  put  in  0.2  mol.  solution 
of  the  same  salt,  nervous  excitability  is  abolished  in  a 
short  time  without  any  primary  stimulation.  The 
carbon  dioxide  production  follows  exactly  analogously  to 
this.  The  nerve  treated  with  the  stronger  solution 
gives  off  more  carbon  dioxide  than  the  one  treated 
with  the  weaker  solution.  This  was  true  even  after 
the  nerve  became  inexcitable,  showing  that  the  nerve 
must  still  be  giving  off  more  carbon  dioxide  while  being 
stimulated  by  the  stronger  solution.  Mr.  Riggs  is 
making  an  extensive  study  of  the  effect  of  various 


CHEMICAL  SIGNS  OF  IRRITABILITY  43 

sddium  salt  solutions  of  varying  concentrations  and  has 
already  discovered  confirmatory  evidence  for  the  increase 
of  metabolism  during  chemical  stimulation.  It  may 
be  added  here  in  passing  that  the  different  solubility  of 
carbon  dioxide  in  these  salt  solutions  cannot  alone  explain 
our  results,  for  there  is  not  enough  difference  in  solu- 
bility of  this  gas  in  such  dilute  equimolecular  solutions 
of  potassium  and  sodium  chloride  whose  effects  on  carbon 
dioxide  production  are  so  divergent,  the  former  salt 
diminishing,  the  latter  increasing,  it. 

The  fact  that  during  chemical  stimulation  the  nerve 
gives  off  more  carbon  dioxide  is  made  evident,  also,  by 
the  use  of  low  concentrations  of  anesthetics.  If  the 
concentration  is  so  low  as  to  give  a  primary  stimulation 
to  the  nerve,  the  production  of  this  gas  is  greatly  acceler- 
ated at  the  beginning  of  immersion  of  the  nerve  in  the 
narcotics.  This  is  an  additional  evidence  that  there 
is  a  relation  between  excitation  and  metabolic  activity. 

Stimulation  in  hydrogen. — The  last  experiment  which 
we  shall  describe  in  this  connection  is  on  the  quantitative 
estimation  of  the  carbon  dioxide  production  in  a  nerve 
when  the  latter  is  in  an  atmosphere  of  hydrogen  and  when 
it  is  being  stimulated  by  an  electrical  current.  We 
expected  to  find  here  one  of  two  things.  First,  there 
is  evidence,  to  which  reference  has  already  been  made, 
that  nerves  left  in  hydrogen  gas  show  diminished  irri- 
tability and  that  they  give  off  smaller  amounts  of  carbon 
dioxide  than  do  the  same  nerves  in  air.  This  fact  led 
us  to  anticipate  that  these  nerves,  being  thus  less  irritable 
than  normal  nerves,  would  produce  less  than  the  usual 
increment  of  carbon  dioxide  on  excitation.  This  would 
be  the  case  if  the  increment  were  a  proper  measure  of  the 


44 


A  CHEMICAL  SIGN  OF  LIFE 


state  of  excitability.  On  the  other  hand,  if  this  carbon 
dioxide  increment  were  not  correlated  with  the  vitality 
of  the  nerve,  and  if  nerve  activity  did  not  involve  respira- 
tion, we  expected  to  find  that  putting  the  nerve  in 
hydrogen  gas  would  not  affect  the  output  on  stimulation. 
For  this  experiment  the  claw  nerve  of  a  spider  crab  was 
used,  and  stimulation  was  effected  in  the  usual  manner 
by  a  tetanizing  induced  current  of  the  same  strength  as 
that  which  had  been  used  before  and  found  to  increase 
the  output  in  normal  nerves.  The  results  are  given  in 
Table  VI.  It  will  be  seen  in  this  table  that  the  pro- 
duction of  carbon  dioxide  by  this  nerve  was  reduced 


TABLE  VI 
COMPARATIVE  RATES  OF  CO*  PRODUCTION  IN  THE  NERVE  WITH  AND  WITHOUT  OXYGEN 


Nerve 

Medium 

Amount  of  COa 
Produced  by 
10  mg.  of  Resting 
Nerve  in  10 
Minutes 

Amount  of  CO, 
Produced  by 
10  mg.  of  Stimu- 
lated Nerve  in 
10  Minutes 

Claw  nerve  of  spider  crab.  . 
Claw  nerve  of  spider  crab  .  . 
Claw  nerve  of  spider  crab.  . 

CO*  free  air 
CO,  free  air 
CO,  free 
hydrogen 

6.  7Xio-7  g.(i5°-i6°) 
7.  gXio-7  g.(20?2) 

3.  4Xio-7  g.(23?o) 

i6Xio-7g.(i4°-i60) 

3.  6XIQ-7  g.(ai?o) 

almost  exactly  50  per  cent  when  the  nerve  was  in  hydro- 
gen, as  compared  with  when  the  nerve  was  in  the  air; 
and  still  more  remarkable  is  the  fact  that  stimulation  in 
the  hydrogen  atmosphere  produced  practically  no 
change  in  the  carbon  dioxide  output.  We  interpret  this 
to  mean  that  the  excitability  of  the  nerve  had  been  so 
reduced  by  the  lack  of  oxygen  that  this  strength  of 
stimulus  was  unable  to  cause  any  excitation  in  the  nerve. 
We  base  this  conclusion  on  the  known  fact  that  lack  of 
oxygen  lowers  considerably  the  irritability  of  the  nerve 


CHEMICAL  SIGNS  OF  IRRITABILITY  45 

and  also  reduces  the  time  during  which  a  current  of  any 
strength  can  stimulate  the  nerve.  Exhaustion  comes 
on  much  more  rapidly  in  a  hydrogen  atmosphere. 
Frohlich  found  that  when  a  sciatic  nerve  of  a  frog  is 
deprived  of  atmospheric  oxygen  its  irritability,  measured 
by  the  threshold  of  stimulation  for  muscular  contraction, 
decreases  more  and  more,  until  after  the  lapse  of  some 
hours  the  stimulation  required  is  so  strong  as  to  approach 
the  region  where  electrical  currents  spreading  down  the 
nerve  stimulate  the  muscle  directly.  If  such  is  the 
case  in  a  frog's  nerve,  the  claw  nerve,  too,  left  in  hydrogen 
may  in  reality  not  be  stimulated  by  such  a  weak  current. 
Thorner,  also,  taking  the  action  current  as  an  index, 
found  that  a  nerve  continuously  stimulated  in  an  atmos- 
phere deficient  in  oxygen  was  quickly  exhausted.  It 
is  remarkable  that  the  action  current  of  a  nerve  in 
nitrogen  gas  falls  to  two-thirds  of  its  original  value 
within  the  first  ten  minutes.  Fatigue  of  the  nerve  by 
continuous  stimulation  during  the  first  few  minutes  of 
our  experiments  with  hydrogen  may  then  have  been 
brought  about. 

Whatever  interpretation  we  take — and,  as  a  matter 
of  fact,  both  factors  doubtless  enter  here — the  fact  that 
there  is  no  decided  increase  of  carbon  dioxide  on  weak 
electrical  stimulation  in  hydrogen  points  inevitably  to 
the  view  that  oxygen  is  a  primary  factor  in  the  excita- 
bility of  the  nerve,  as  well  as  in  the  conduction  of  the 
nerve  impulse. 

Recently  Bayliss  has  pointed  out  what  he  considers 
a  probable  error  in  our  experiments.  To  him  it  seems 
that  the  increased  production  of  carbon  dioxide  on 
electrical  stimulation  may  be  due,  in  consequence  of  the 


46  A  CHEMICAL  SIGN  OF  LIFE 

heat  produced  by  the  exciting  current  itself,  to  the 
escape  of  carbon  dioxide  which  had  been  dissolved  in 
the  living  cells  in  the  connective  tissue  around  the  nerve 
fiber.  We  have  cited  several  experiments  the  results  of 
which  exclude  this  possibility.  In  addition  to  these, 
the  apparent  lack  of  any  increase  of  this  gas  on  applica- 
tion of  induction  shocks  to  a  nerve  in  an  oxygen-free 
medium  like  hydrogen  should  be  taken  as  conclusive  evi- 
dence that  the  increased  gas  production  by  a  nerve  when 
stimulated  in  the  air  is  due  to  physiological  processes,  and 
not  to  experimental  errors. 

w/  Lack  of  fatigue. — If  the  chemical  change  of  the  nerve 
tissue  is  as  active  as  the  observations  just  cited  indicate, 
one  naturally  asks  how  we  can  explain  the  fact  that  the 
nerve  impulse  can  pass  continuously  through  the  fiber 
without  any  measurable  sign  of  fatigue.  There  is  no 
doubt  that  this  apparent  lack  of  fatigue  of  medullated 
nerves  is  a  very  remarkable  and  striking  phenomenon. 
Nerves  can  be  stimulated  for  many  hours  continuously 
without  marked  fatigue.  But  it  does  not  at  all  mean 
that  there  is  no  chemical  change  in  the  nerve,  for,  in 
the  first  place,  it  must  not  be  forgotten  that  medullated 
nerves  have  in  the  medullary  sheath  a  very  large  supply 
of  raw  material,  or  food,  which  is  more  than  sufficient  for 
their  nutrition  during  the  longest  experiments  which  have 
been  tried.  The  only  surprising  feature  of  the  physiology 
of  the  nerve  is  that  in  the  isolated  nerve,  where  there  is 
no  opportunity  for  getting  rid  of  the  products  of  decom- 
position, accompanying  functional  activity,  by  way  of 
the  blood,  nevertheless  these  products  do  not  seem  to 
act  deleteriously  on  the  nerve  function.  But,  after  all, 
we  have  only  to  assume,  in  order  to  understand  this,  that 


CHEMICAL  SIGNS  OF  IRRITABILITY  47 

tKese  products  are  of  such  a  nature  that  they  have  very 
little  physiological  action.  It  is  quite  possible  that 
they  are  taken  care  of  in  the  nerve,  because  it  is  vitally 
necessary  to  animals  that  those  of  their  nerves  which 
go  to  skeletal  muscle,  at  any  rate,  shall  not  be  easily 
fatigued.  There  are  also  other  tissues  in  which  it  is 
perfectly  certain  that  there  is  a  rapid  metabolism  and 
which  also  show  a  no  less  remarkable  freedom  from 
fatigue.  We  may  cite,  for  example,  the  contracting 
wings  of  insects  which  vibrate  at  a  rate  as  high  as  three 
hundred  vibrations  per  second,  and  yet  these  insects  can 
fly  for  hours  continuously  without  muscular  fatigue. 
There  is  not  the  least  doubt  that  these  muscles  which 
are  undergoing  this  tremendous  activity  without  fatigue 
are  at  the  same  time  undergoing  a  very  rapid  metabolism. 
All  that  is  necessary  to  avoid  fatigue  is  that  the  tissue 
shall  return  each  time  after  activity  to  its  normal  state. 
The  ordinary  induction  coil  which  we  use  in  these  experi- 
ments only  stimulates  a  nerve  about  one  hundred  times 
a  second,  or  about  one-third  as  often  as  the  insect's  wing 
muscles  contract,  so  that  more  time  is  given  for  recovery 
in  the  nerve  than  in  these  muscles.  The  lack  of  appar- 
ent fatigue  in  nerves  is  not,  then,  any  proof  of  the 
absence  of  metabolism. 

When  we  examine  nerves  more  closely  and  by  more 
delicate  methods,  we  find  unmistakable  evidences  of 
fatigue  in  them.  The  only  remarkable  thing  about  them 
is  that  they  recover  from  that  fatigue  very  rapidly. 
Thus  Gotch  and  Burch  discovered  in  1889  that  if  two 
stimuli  are  successively  applied  to  a  nerve  within 
1/5,000  of  a  second,  only  a  single  nerve  impulse  is  pro- 
duced. One  cannot  generate  a  second  impulse  until 


48  A  CHEMICAL  SIGN  OF  LIFE 

the  nerve  has  recovered  from  the  first.  This  refractory 
period  of  1/5,000  of  a  second  may  be  considerably  pro- 
longed under  certain  conditions,  such  as  low  temperature, 
high  temperature,  asphyxiation,  various  drugs,  and 
certain  anesthetics.  Frolich  prolonged  this  refractory 
period  by  partial  anesthesia  and  succeeded  in  producing 
fatigue  phenomena  by  repeated  electrical  stimulations 
at  shorter  intervals  than  the  prolonged  refractory  period 
of  the  nerve. 

The  idea  that  all  the  physiological  activities  are 
composed  of  at  least  two  opposing  metabolic  phenomena 
was  expressed  by  Claude  Barnard  and  later  extended  by 
Hering.  Thus  metabolic  activities  are  considered  as 
consisting  of  two  phases,  namely,  a  breaking  down,  or 
katabolic,  and  a  building  up,  or  anabolic,  phase.  That 
two  such  phenomena  are  involved  in  nervous  metabolism 
and  are  closely  connected  with  the  phenomena  of  fatigue 
may  be  shown  by  the  use  of  certain  drugs  in  connection 
with  electrical  changes  and  refractory  periods.  Waller 
observed  that  protoveratrin  slows  up  one  of  the  electrical 
changes  (positive  variation)  of  the  nerve,  while  the 
other  (negative  variation)  is  little  influenced.  He 
contended  accordingly  that  this  drug  does  not  alter 
katabolic  changes  of  the  nervous  metabolism  but  re- 
tards the  anabolic  activity  to  a  considerable  degree.  It 
is  by  its  anabolism  that  the  nerve  is  restored  to  its  nor- 
mal state  after  the  passage  of  the  impulse.  Since  the 
pharmacological  action  of  protoveratrin  and  yohimbin 
on  muscle  are  known  to  be  very  similar,  Tait  concludes 
from  the  study  of  the  effect  of  yohimbin  on  the  refractory 
period  of  the  nerve  that  these  drugs  must  attack  nerves 
in  a  similar  manner.  Yohimbin,  in  other  words,  retards 


CHEMICAL  SIGNS  OF  IRRITABILITY  49 

anabolic  processes  considerably,  thus  prolonging  the  one 
phase  of  the  refractory  period  or  increasing  thus  the 
inefficiency  of  the  nerve.  From  these  considerations  we 
may  conclude  that  the  nerve  may  be  fatigued  by  repeated 
stimulations  if  we  can  prolong  the  time  interval  of  either 
the  excitatory  or  the  repair  state. 

The  general  conclusion  to  which  this  leads  us  is  that 
what  we  call  fatigue  in  a  tissue  of  any  kind  is  due  to  a 
failure  of  the  tissue  to  recover  completely  its  normal 
state  after  it  is  excited.  In  some  tissues  this  state  of 
fatigue  is  very  easily  demonstrated,  but  in  medullated 
nerves  the  mechanism  of  recovery  is  so  perfect  that 
ordinarily  the  restoration  of  the  nervous  substance  to  its 
original  state  after  the  passage  of  the  impulse  takes  a 
very  short  time — -a.  fraction  of  a  thousandth  of  a  second. 
Nevertheless,  by  the  conditions  stated,  namely,  by  lack 
of  oxygen,  by  partial  anesthetization,  by  the  action  of 
drugs  like  yohimbin  and  protoveratrin,  the  recovery  is 
delayed,  and  in  these  cases  the  nerve  exhibits  phenomena 
which  may  properly  be  called  fatigue.  The  failure  of  a 
nerve  to  show  fatigue  under  ordinary  circumstances 
should  not,  therefore,  cause  us  to  conclude  on  this 
account  that  there  had  been  no  destruction  of  nerve 
substance  by  its  excitation,  but  rather  that  the  nerve 
had  in  its  medullary  sheath  an  especial  supply  of  a  food 
particularly  formed  to  serve  as  a  speedy  pabulum  for 
the  fibers,  and  that  the  means  of  reconstituting  the 
nerve  tissue  after  excitation  had  been  so  perfected  that 
the  result  was  accomplished  in  a  very  brief  time. 

Heat  formation. — Another  evidence  which  has  been 
often  cited  as  showing  that  there  was  no  chemical  change 
accompanying  the  nerve  excitation  is  the  fact  that  there 


50  A  CHEMICAL  SIGN  OF  LIFE 

is  no  heat  produced  in  excited  nerves.  How  shall  we 
explain  the  fact  that  this  relatively  tremendous  chemical 
transformation  can  occur  without  heat  formation? 
There  are  several  explanations  which  might  be  given 
of  this  fact,  but  before  considering  them  we  may  see 
first  what  the  evidence  is  that  there  is  no  heat  produced. 

Although  there  have  been  in  the  literature  many 
contradictory  statements  as  to  heat  formation  in  the 
active  nerve,  the  original  negative  results  of  Helmholz, 
Stewart,  and  Rolleston  have  been  confirmed  recently 
by  A.  V.  Hill's  work,  which  shows  that  there  is  no  meas- 
urable liberation  of  heat  when  the  nerve  is  stimulated. 
Since  his  apparatus  is  exceedingly  sensitive,  being  sus- 
ceptible to  the  change  of  1/1,000,000  of  a  degree  Centi- 
grade, the  lack  of  observed  heat  production  is  not  ap- 
parently to  be  explained  by  any  lack  of  a  proper  method 
of  measuring  temperatures.  His  work  is  remarkably 
significant  in  that  according  to  his  calculation  not  more 
than  one  single  oxygen  molecule  in  every  cube  of  a 
nerve  containing  3 . 7  cubic  /-t  can  be  used  up  by  a  single 
propagated  nerve  impulse,  since  more  than  this  amount 
would  produce  a  measurable  amount  of  heat.  Thus  he 
is  convinced  that  a  nerve  impulse  is  not  of  an  irreversible 
chemical  nature,  but  must  be  of  a  purely  physical  nature. 

Negative  evidence  of  this  kind  cannot  be  taken  at. 
its  face  value  without  considering  the  limitations  of  the 
method.  Stewart  calls  attention  to  the  fact  that  we 
should  not  forget  that  if  the  axis  cylinder  is  the  only 
portion  which  is  conducting  a  nerve  impulse,  as  we 
believe,  the  measurement  in  medullated  nerves  with 
which  most  experiments  were  made  does  not  express 
the  true  state  in  the  axis  cylinder.  We  should  consider, 


CHEMICAL  SIGNS  OF  IRRITABILITY  51 

not'  only  the  volume  ratio  between  the  axis  cylinder 
and  sheath,  but  also  the  exact  coefficient  of  the  tempera- 
ture radiation  in  the  sheath.  In  this  connection  it  is 
interesting  to  note  the  very  recent  work  of  Snyder,  who 
showed  that  a  smooth  muscle  also,  when  measured 
in  the  same  way,  failed  to  produce  heat  during  its  con- 
traction. But  no  one  doubts  that  during  muscle  work 
the  metabolic  activity  is  greatly  accelerated.  He  used, 
by  the  way,  exactly  the  same  technique  as  Hill.  Either, 
then,  heat  is  produced,  but  owing  to  some  circumstance 
it  is  not  detected,  or  else  it  is  not  produced.  Now,  most 
muscles  certainly  produce  heat  when  they  work,  and 
it  is  probable  that  smooth  muscle  does  so  also.  It  is 
not  to  be  supposed  that  the  muscles  are  a  perfectly 
reversible  engine.  On  the  contrary,  we  all  know  that 
we  become  warm  when  we  exercise.  When,  then,  it  is 
reported  that  smooth  muscle  produces  no  heat  when  it 
contracts,  we  are  at  once  skeptical  of  the  method  which 
gives  such  a  result.  Consequently,  therefore,  while  the 
method  for  the  detection  of  the  nerve  heat  on  stimula- 
tion appears  to  be  a  competent  method,  we  do  not  feel 
certain  that  this  is  the  case. 

But  suppose  we  grant  that  the  results  are  correct — 
that  nerves  produce  no  heat  when  they  are  excited — 
does  that  mean  that  there  is  no  chemical  change  occur- 
ring in  the  nerve  ?  Is  this  fact  conclusive  evidence  that 
these  results  of  a  positive  kind  which  we  have  adduced, 
showing  that  chemical  changes  do  occur  in  the  nerves, 
are,  after  all,  due  to  some  secondary  cause  or  to  some 
undiscovered  errors  of  technique  on  our  part?  Cer- 
tainly this  is  not  the  case,  for  it  is  quite  possible  for 
chemical  changes  to  occur  without  liberating  more  than 


52  A  CHEMICAL  SIGN  OF  LIFE 

a  very  small  amount  of  heat,  or,  indeed,  they  may 
actually  be  heat-consuming  rather  than  heat-producing. 
And,  indeed,  if  we  had  side  by  side  reactions  which  pro- 
duce and  reactions  which  consume  heat  we  might  have  a 
considerable  chemical  change  without  the  liberation  of 
much  heat.  Thus  in  a  Daniels'  cell  there  is  a  very 
large  transformation  of  energy  with  the  liberation  of 
very  little  heat.  The  Weston  cell  has  a  still  smaller 
heat  coefficient.  The  energy  set  free  in  the  cell  takes 
the  form  of  electrical  energy  rather  than  heat.  To  be 
sure,  it  is  ultimately  converted  into  heat,  but  for  the 
time  being  it  does  not  appear  as  such.  There  are  many 
chemical  changes  also  which  yield  carbon  dioxide  and 
yet  liberate  very  little  heat.  It  is  possible  that  the 
carbon  dioxide  is  not  produced  by  an  oxidation,  but  by 
a  fermentative  process  which  is  hardly  exothermic. 
Many  such  hydrolyses  liberate  almost  no  heat  at  all. 
We  might  have,  for  example,  the  oxidation  going  on  at  a 
steady  rate  all  the  time,  independently  of  the  stimulus. 
By  this  means  a  constant  production  of  heat  occurs,  but 
carbon  dioxide  is  not  liberated.  That  is,  the  change 
has  occurred  at  a  steady  rate  in  the  oxygen  atoms,  which 
is  the  essence  of  the  oxidation.  A  very  unstable  com- 
pound might  result,  awaiting  only  the  hydrolysis  of  the 
carbon  dioxide.  This  last  process  might  be  that  which 
is  accelerated  by  the  stimulation  and  the  passage  of  the 
impulse.  This  liberates  gas,  but  very  little  heat.  The 
reconstitution  of  the  irritable  substance  might  then  be 
brought  about  by  a  second  molecule  slipping  in  to  take 
the  place  of  the  first,  while  the  exhausted  molecule  was 
withdrawn  to  be  reoxidized  and  thus  made  ready  for  use 
again.  This  reoxidation  perhaps  goes  on  all  the  time, 


CHEMICAL  SIGNS  OF  IRRITABILITY  53 

irrespective  of  the  stimulus,  and  accordingly  excitation 
appears  to  generate  no  heat  and  there  appears  to  be  no 
fatigue,  unless  we  deprive  the  nerve  of  oxygen  for  some 
time,  and  yet  we  have  a  copious  production  of  carbon 
dioxide  which  is  increased  on  stimulation.  The  fact, 
therefore,  that  there  is  no  increased  heat  production  in  a 
stimulated  nerve  is  by  no  means  contrary  to  our  results, 
although  it  is  certainly  surprising.  It  indicates,  perhaps, 
that  the  act  of  excitation  is  not  primarily  an  oxidation, 
but  that  the  oxidation  is  concerned  in  the  processes  of 
repair.  There  are  several  facts  which  might  be  cited, 
were  the  space  at  our  command,  which  would  lead  to  the 
same  conclusion.  There  are  also  other  suggestions  which 
might  be  made  to  account  for  this  seeming  incompatibil- 
ity, but  it  would  be  useless  to  do  so  without  experiments. 

We  may  therefore  close  this  brief  discussion  with  the 
statement  that  the  failure  to  detect  heat  production  in 
nerves  during  excitation  is  no  evidence  of  value  against 
the  occurrence  there  of  chemical  changes  resulting  in 
carbon  dioxide  production  and  correlated  with  the  irri- 
tability. The  conclusion  drawn  from  it  by  some  authors 
that  the  nerve  impulse  does  not,  on  this  account,  involve 
any  chemical  processes  is  entirely  unwarranted. 

We  may  in  this  connection  stop  for  a  moment  to 
consider  what  is  known  of  the  oxygen  consumption  of 
nerves,  for  while  we  have  ourselves  as  yet  carried  out 
no  experiments  in  this  line,  yet  there  have  been  some 
observations  made  which  can  be  correlated  with  the 
carbon  dioxide  production.  In  the  first  place,  it  may 
be  noted  that  there  is  no  immediate  dependence  of  some 
nerves,  at  least,  on  atmospheric  oxygen  for  their  activity. 
In  this  respect  the  carbon  dioxide  production , and  the 


54  A  CHEMICAL  SIGN  OF  LIFE 

oxygen  consumption  of  the  nerve  appear  to  be  on  a 
somewhat  different  footing.  I  say  "appear  to  be," 
because  the  methods  of  determining  the  oxygen  con- 
sumption are  still  rather  crude,  and  the  studies  have 
been  few.  A  nerve  always  gives  off  more  carbon  dioxide 
when  it  is  stimulated  or  active,  whereas  we  know  very 
little  about  whether  its  intake  of  oxygen  is  increased 
in  anything  like  the  same  degree.  The  sciatic  nerve 
of  a  frog — a  medullated  nerve — can  remain  excitable 
for  a  long  time  in  the  absence  of  atmospheric  oxygen, 
although  its  irritability  diminishes  under  these  cir- 
cumstances, and,  as  already  explained,  its  fatigability 
increases. 

There  is  a  considerable  amount  of  evidence  to  show 
that  oxygen  is  very  closely  associated  with  the  state  of 
excitability.  To  harmonize  these  two  facts,  namely, 
the  independence  of  atmospheric  oxygen  and  the  fact 
just  stated,  the  oxygen-storage  hypothesis  has  been 
suggested,  by  which  the  exhaustion  is  attributed  to 
complete  consumption  of  stored  oxygen.  Excitability 
is  restored  when  atmospheric  oxygen  is  readmitted. 
Without  committing  ourselves  to  this  hypothesis,  we  may 
add  that  according  to  Haberlandt's  figure  the  resting 
nerve  of  10  mg.  weight  will  consume  only  0.0042  c.c. 
oxygen  in  ten  hours.  If  we  take  our  figure  of  carbon 
dioxide  output  and  assume  that  one  volume  of  oxygen 
was  necessary  to  produce  one  volume  of  carbon  dioxide 
(this  assumption  is  made  without  any  significance 
except  to  give  a  liberal  estimate),  the  carbon  dioxide 
production  would  require  a  consumption  of  about 
0.015  c.c.  of  oxygen  for  ten  hours.  And  if  we  assume 
again  that  activity  will  increase  oxygen  consumption 


CHEMICAL  SIGNS  OF  IRRITABILITY  55 

in'  proportion  to  carbon  dioxide  production,  then  it 
means  that  the  nerve  when  stimulated  would  take  up 
only  0.03  c.c.  of  oxygen  during  ten  hours'  stimulation. 
It  is  extremely  difficult,  as  everyone  who  has  tried  it 
knows,  to  free  any  gas  from  such  small  amounts  of 
oxygen  as  those  which  are  required  to  keep  up  irrita- 
bility. Our  experience  in  freeing  gases  from  traces  of 
carbon  dioxide  makes  us  realize  the  difficulty  of  getting 
the  nerve  in  the  first  place  in  a  gas  quite  free  from 
oxygen,  and  we  believe  that  many  experiments  have 
been  tried  in  which  there  is  still  some  probability  that 
enough  oxygen  remained  to  supply  these  small  amounts 
needed.  More  delicate  determinations  will  have  to 
be  made  before  we  feel  certain  that  nerves  have  been 
found  to  be  irritable  for  some  time  in  atmospheres  which 
are  free  beyond  question  from  all  traces  of  oxygen.  How 
shall  we  know  when  the  gas  we  use  is  free  from  oxygen 
in  these  minute  amounts  ?  Yet  until  we  know  this  it  is 
impossible  to  study  accurately  the  relation  of  irritability 
to  oxygen.  Meanwhile,  however,  we  may  recall  the 
fact  that  carbon  dioxide  production  in  the  spider  crab's 
nerve  is  not  only  reduced  in  the  absence  of  oxygen, 
but  also  that  we  cannot  increase  its  production  in  such 
an  atmosphere  by  a  stimulation  which  in  the  presence 
of  oxygen  increased  the  production  of  carbon  dioxide 
over  200  .per  cent.  These  facts  show  conclusively, 
negative  evidence  to  the  contrary  notwithstanding,  that 
oxygen  is  in  some  way  involved  in  the  anabolism  or 
katabolism  of  nerve  fibers. 

Summary. — The  facts  presented  in  this  chapter 
prove  that  all  kinds  of  nerves,  medullated  and  non- 
medulla  ted,  when  stimulated  increase  their  output  of 


56  A  CHEMICAL  SIGN  OF  LIFE 

carbon  dioxide  two  or  three  times  above  that  of  their 
resting  state.  This  increase  comes  whatever  the  method 
of  stimulation,  provided  only  that  the  nerve  is  alive 
and  irritable.  Dead  or  anesthetized  nerves  show  no 
such  an  increase.  The  state  of  excitation  in  a  nerve, 
which  we  call  a  nerve  impulse  when  it  spreads  from  one 
place  to  another,  is  not  a  purely  physical  change  of 
state,  as  it  has  been  represented  hitherto  as  being,  but  it 
undoubtedly  involves  a  corresponding  chemical  change. 
Perhaps  the  excitation  is  this  chemical  change  itself. 
Furthermore,  the  facts  that  nerves  do  not  increase 
their  heat  output  on  stimulation  and  that  they  are  nearly 
free  from  fatigue  effects  are  evidently  not  incompatible 
with  the  vigorous  metabolism  discovered  to  exist  in  them. 


CHAPTER  IV 
EXCITATION  AND  CONDUCTION 

We  have  shown  by  this  study  of  nerves  that  living 
matter  must  necessarily  undergo  metabolic  activity  and 
that  without  an  increase  of  this  activity  protoplasm 
will  not  function.  In  short,  to  be  excitable,  the 
protoplasm  must  respire,  and  to  be  excited,  its  meta- 
bolic activity  must  be  accelerated.  It  has  also  been 
demonstrated  that  the  excited  state  travels  along  the 
fiber  with  simultaneous  increase  of  the  metabolism. 
Although  our  theme  in  this  little  volume  is  not  a 
consideration  of  how  this  state  of  excitation  is  trans- 
mitted, but  is  rather  an  analysis  of  the  conditions 
which  characterize  the  irritable  tissue,  the  relation 
between  these  two  phenomena  is  so  close  that  we 
shall  consider  certain  facts  which  are  directly  con- 
cerned with  them. 

The  two  phases  of  protoplasmic  irritability  are 
excitability  and  conductivity,  or  transmission,  of  this 
excitation.  Since  it  is  very  difficult  experimentally  to 
produce  excitation  without  conduction,  we  are  accus- 
tomed to  consider  the  fundamental  processes  underlying 
these  two  processes  as  probably  identical.  There  .are 
certain  facts  which  are  sometimes  cited  as  evidence  that 
these  phenomena  are  not  necessarily  interdependent. 
In  the  case  of  localized  and  partial  narcosis^  for  instance, 
local  excitability  in  the  narcotized  portion  does  not 
disappear  simultaneously  with  conductivity  through 

57 


58  A  CHEMICAL  SIGN  OF  LIFE 

this  region,  assuming,  of  course,  that  the  same  strength 
of  current  when  stimulating  in  or  above  the  narcotized 
part  is  made  use  of.  Since  we  have  no  evidence  that  the 
resistance  of  the  surrounding  sheath  of  the  fiber  and 
that  of  the  conducting  medium  are  the  same,  we  cannot 
assume  that  in  both  experiments  the  same  strength 
of  stimulus  was  really  applied  to  the  conducting  portion. 
The  non-transmissibility  of  the  inhibitory  state  is 
regarded  as  another  distinction  between  excitation  and 
conduction.  We  can  abolish  excitability  at  one  point 
without  making  its  neighboring  region  inexci table.  It 
is  rather  difficult  to  consider  an  analogy  between  depres- 
sion and  excitation,  but  the  fact  is  -that  even  if  we  may 
not  be  able  to  make  other  than  one  point  inexcitable 
by  one  depressing  agent,  it  is  doubtful  whether  we  can 
produce  local  inexcitability  without  affecting  the  con- 
tiguous parts  of  the  nerve.  Waller  has  demonstrated, 
in  the  case  of  inhibition  by  heat,  that  the  point  of 
application  of  gentle  heat  became  electropositive  to  the 
rest  of  the  nerve  instead  of  negative,  as  is  the  case  in 
ordinary  stimulation.  According  to  him,  heat  does  not 
stimulate  the  tissue,  but  depresses  it.  If  this  is  the 
case,  as  he  seems  to  have  demonstrated  in  a  variety  of 
tissues,  it  indicates  that  although  we  cannot  produce 
depression  at  points  other  than  the  point  of  application, 
yet  certain  conditions  along  the  nerve  must  surely  be 
altered  through  such  an  inhibition.  In  any  event,  we 
cannot  consider  non-conductivity  of  the  inhibitory  state 
as  evidence  that  excitability  and  conductivity  are 
entirely  different  processes. 

Let  us  now  consider  in  detail  the  relation  between 
excitation  and  conduction. 


EXCITATION  AND  CONDUCTION  59 

1  Excitability. — The  excitability  of  the  nerve  fiber  has 
three  criteria:  (i)  the  degree  of  irritability,  i.e.,  the 
ease  with  which  it  can  be  stimulated;  (2)  velocity  of 
the  nerve  impulse,  i.e.,  the  speed  with  which  the  state 
of  excitation  travels  from  one  point  to  another;  (3) 
the  direction  of  the  nerve  impulse.  All  nerves  are 
classified  into  two  general  functional  types:  efferent 
and  afferent,  the  former  conducting  away  from  the 
nerve  center  (brain,  etc.),  the  latter  toward  the  center. 
We  shall  consider  somewhat  in  detail  in  this  chapter 
what  relation  the  metabolic  condition  bears  to  these 
three  phenomena  in  the  nerve. 

Degree  of  excitability. — Not  all  nerves  can  be  stimu- 
lated equally  well  by  the  same  strength  of  stimulus. 
The  threshold  value — the  minimum  strength  of  stimulus 
which  can  call  forth  functional  activity — is  different 
in  different  nerves.  Not  only  have  the  different  nerves 
different  degrees  of  excitability,  but  the  same  nerve  can 
be  made  excitable  in  different  degrees  under  a  variety  of 
conditions.  If  we  study  metabolic  activity  in  nerves 
under  different  conditions  which  we  know  affect  the 
state  of  excitability,  we  find  that  there  is  a  very  close 
relation  between  metabolism  and  excitability. 

a)  If  the  sciatic  nerve  is  removed  from  a  frog,  it 
exhibits  electrical  phenomena  for  many  hours.  Since 
electrical  changes  are  characteristic  of  living  nerves 
only,  we  consider  that  the  isolated  nerve  does  not  die  for 
many  hours.  Such  a  nerve,  although  it  shows  large 
electrical  responses,  is  nevertheless  less  excitable  than  a 
fresh  one.  If  measurements  are  made  on  an  isolated 
nerve  at  successive  time  intervals  for  many  hours,  we 
find  that  the  carbon  dioxide  production  steadily  dimin- 


60  A  CHEMICAL  SIGN  OF  LIFE 

ishes  as  the  nerve  approaches  death.  The  point  of 
minimum  production  of  the  gas  corresponds  approxi- 
mately to  the  point  where  an  electrical  response  ceases 
(see  p.  28). 

b)  Although  the  nerve  remains  active  for  some  time 
without  oxygen,  it  is  known  that  the  absence  of  oxygen 
diminishes  the  excitability  of  the  nerve.     This  diminu- 
tion of  the  excitability  when  in  hydrogen  is  accompanied 
with  a  lowering  of  carbon  dioxide  production  in  the  nerve. 

c)  Further    facts    showing    the    relation    between 
excitability  and  metabolic  activity  are  brought  out  by 
the  study  of  the  effects  of  narcotics  on  the  nervous 
metabolism.     There  are  several  compounds  which  alter 
the  state  of  excitability  of  nerves  to  a  considerable 
degree.     The  discovery  of  just  what  happens  to  respira- 
tion during  anesthesia  will  throw  much  light  on  the 
nature  of  irritability.     It  is  this  which  we  shall  now 
study  in  detail. 

In  recent  years  many  experiments  have  been  per- 
formed which  are  supposed  to  prove  that  oxygen  con- 
sumption can  go  on  uninterruptedly  during  narcosis,  and 
the  consequent  conclusion  has  been  that  narcosis  is  not 
produced  by  asphyxiation.  This  is  not  the  place  for  us 
to  weigh  the  merit  of  these  arguments,  nor  are  we  con- 
cerned here  with  the  question  of  how  narcotics  act  on 
protoplasm,  but  it  is  very  important  to  know  whether  or 
not  metabolic  activity  in  nervous  tissue  can  go  on  undis- 
turbed while  the  tissue  is  unable  to  perform  its  own 
function.  Are  respiration  and  irritability  independent 
processes  ?  To  show  that  they  are  dependent  we  shall 
cite  in  detail  experiments  on  the  effects  of  anesthetics  on 
respiration. 


EXCITATION  AND  CONDUCTION  61 

*  We  have  already  mentioned  the  effect  of  ether  on 
nerves  and  demonstrated  that  lowering  the  excitability  of 
a  nerve  is  accompanied  by  a  lowering  of  carbon  dioxide 
production.  For  the  quantitative  experiments,  carried 
out  in  conjunction  with  Dr.  Adams,  we  used  chloral 
hydrate  and  ethyl  urethane  in  preference  to  the  ordinary 
volatile  narcotics.  If  we  anesthetize  a  nerve  with  the 
lowest  concentration  of  ether  or  chloroform  that  pro- 
duces a  reversible  loss  of  irritability,  then  the  anesthe- 
tized nerve  regains  its  excitability  during  the  course  of  the 
experiments,  for  in  order  to  make  the  apparatus  free 
from  carbon  dioxide  after  introducing  the  nerve  we  have 
to  wash  it  with  carbon-dioxide-free  air  several  times. 
By  so  doing  the  most  volatile  narcotics  are  removed 
from  the  nerve.  On  the  other  hand,  if  we  use  higher 
concentrations,  which,  as  we  know,  lower  carbon  dioxide 
production,  we  may  be  subject  to  the  criticism  that  the 
lowering  of  metabolism  may  be  due  partly  to  death  or 
injury.  It  is  therefore  essential  that  we  should  investi- 
gate the  effect  of  various  concentrations,  from  such  as 
have  apparently  no  narcotic  effect  to  those  from  which 
recovery  is  doubtful  or  absent.  Thus  the  use  of  suitable 
narcotics  as  well  as  concentrations  seems  to  be  of  prime 
importance.  For  even  those  who  consider  that  narcosis 
is  not  due  to  an  asphyxiation  admit  that  the  oxygen 
consumption  is  greatly  depressed  if  the  narcosis  is  pushed 
too  far,  although  such  depression  in  the  rate  of  oxidation 
may  have  nothing  to  do  with  the  cause  of  the  narcosis. 

With  a  view  to  studying  the  effect  of  various  con- 
centrations of  anesthetics,  the  claw  nerve  of  a  crab  was 
isolated,  its  excitability  tested  by  electrical  stimulation, 
and,  without  being  cut  off  from  the  claw,  it  was  immersed 


62 


A  CHEMICAL  SIGN  OF  LIFE 


in  the  narcotic  solution,  care  being  taken  that  the  claw 
muscle  did  not  come  in  contact  with  the  solution.  After 
ten  minutes  it  was  removed,  freed  from  excess  liquid  by 
means  of  filter  paper,  its  state  of  excitability  determined 
by  stimulation,  and,  with  claw  attached,  it  was  placed 
in  a  moist  chamber  for  ten  minutes,  which  is  the  time 

TABLE  VII* 
EFFECTS  OF  ETHYL  URETHANE  ON  CLAW  NERVE  OF  SPIDER  CRAB,  Libinia  canaliculate, 


TREATED  BY 

EFFECTS  ON 
EXCITA- 
BILITY 

AFTER 
RETURN  TO 
SEA-WATER 

CHANGE 
IN  WEIGHT 

IN    10 

MINUTES 

AMOUNT  OF  COa  PRO- 
DUCED BY  10  MG.  OF 

NERVE  IN  10  MINUTES 

Concen- 
tration in 

For 
How 

Sea-Water 

Long 

i,  Excitable 

No  change 

7.gXio~7g.  at  20?  2 

o  per  cent  .  . 

10  min. 

nerve 

2,  Inexcit- 

Excitable 

No  change 

5.7X10—7  g.at22° 

able  nerve 

i  per  cent  .  . 

10  min. 

Excitable 

Excitable 

No  change 

2i.7Xio~7  g.at23?8 

2  per  cent  .  . 

10  min. 

Narcosis 

Excitable 

No  change 

Not  determined 

very  slow 

3  percent.  . 

10  min. 

Slow,  partial 

Excitable 

No  change 

Not  determined 

narcosis 

4  percent.. 

10  min. 

Practically 

Good  return 

No  change 

3.3X10"?  g.at2i°-2i?s 

narcotized 

S  percent.  . 

10  min. 

Completely 

Recovery  is 

No  change 

Not  determined 

narcotized 

not  always 

good 

*  Since  our  previous  determinations  of  the  carbon  dioxide  production  of  the  spider 
crab's  nerve  were  made  at  a  much  lower  temperature  (15°  to  16°  C.),  the  work  was 
repeated  at  the  higher  temperature  at  which  most  of  the  present  experiments  were  made. 
In  order  to  make  the  comparison  a  rigid  one,  the  normal  nerve  was  subjected  to  a  treat- 
ment similar  to  that  employed  with  the  narcotized  nerve,  except  that  it  was  not  narcotized. 
It  was  isolated,  quickly  weighed,  and  immersed  in  sea-water  for  ten  minutes,  after  which 
the  rate  of  carbon  dioxide  production  was  determined  in  the  usual  way.  As  was  expected, 
the  nerve  exhibited  a  somewhat  higher  rate  of  metabolism  at  the  higher  temperature. 
The  results  are  incorporated  in  the  table. 

usually  required  in  making  a  determination  of  the 
carbon  dioxide  production.  After  this  the  nerve  was 
brought  back  to  fresh  sea-water  and  the  return  of  irrita- 
bility was  determined,  as  evidenced  by  contraction  of  the 
claw  or  joint  in  response  to  the  electrical  stimulation 
of  the  nerve.  Thus  the  essential  conditions  obtaining 


EXCITATION  AND  CONDUCTION  63 

in   the  actual   determination  of   carbon  dioxide  were 
reproduced. 

From  the  results  so  obtained  the  minimum  concen- 
tration which  produced  a  reversible  loss  of  irritability  was 
chosen  for  our  experiments  on  the  carbon  dioxide  pro- 
duction, and  we  are  thus  assured  that  the  nerve  has  been 
narcotized,  but  that,  since  its  excitability  returns,  no 
permanent  injury  has  been  caused.  The  carbon  dioxide 
production  of  the  nerve  thus  treated  has  been  determined 
and  compared  with  that  of  a  normal  nerve.  These 
results  are  tabulated  with  the  physiological  data  and 
given  in  Table  VII. 

ETHYL  URETHANE 

As  shown  by  physiological  tests,  a  freshly  isolated 
claw  nerve  on  immersion  in  a  4  per  cent  solution  of  ethyl 
ure thane  loses  its  excitability  within  ten  minutes.  Such 
a  nerve,  however,  if  left  in  a  moist  chamber  for  ten  or 
fifteen  minutes  and  then  returned  to  sea-water,  comes 
back  to.  a  normal  condition  of  excitability  with  appar- 
ently no  injurious  effects.  That  the  nerve  so  narcotized 
gives  off  less  carbon  dioxide  than  a  normal  one  can  be 
demonstrated  qualitatively  as  follows : 

Two  nerves  of  approximately  the  same  weight  are 
isolated,  and  one  is  immersed  in  sea- water  while  the 
other  is  treated  with  a  4  per  cent  urethane  solution  for 
ten  minutes.  At  the  end  of  this  time  their  rates  of 
carbon  dioxide  production  are  compared  simultaneously 
in  the  biometer  by  placing  the  normal  nerve,  for  example, 
in  the  right  chamber  and  the  other  in  the  left.  Within 
ten  minutes  the  difference  in  carbon  dioxide  output 
will  become  evident,  for  not  only  does  the  precipitate 


64  A  CHEMICAL  SIGN  OF  LIFE 

appear  first  on  the  barium  hydroxide  in  the  right  cham- 
ber, containing  the  normal  nerve,  but  the  amount  of 
precipitate  later  is  seen  to  be  much  greater  in  this 
chamber  than  in  the  other.  The  narcotized  nerve  is 
giving  off  less  carbon  dioxide  than  the  normal  one. 

That  the  narcotized  nerve  produces  less  carbon  dioxide 
than  the  normal  is  shown  more  strikingly  by  quantita- 
tive determinations.  The  average  carbon  dioxide  output 
for  the  nerve  when  treated  for  ten  minutes  with  a  4 
per  cent  ethyl  urethane  solution  is  less  than  50  per  cent 
of  that  of  the  normal  nerve.  At  20°  to  22°  C.  the  narcot- 
ized nerve  gives  3.3 Xio~7  g.  per  centigram  of  tissue  for 
ten  minutes'  respiration,  while  the  normal  nerve,  calcu- 
lated for  the  same  units,  produces  y.pXio"7  g.  One 
exception  may  be  noted  here — an  experiment  in  which 
the  respiration  of  the  narcotized  nerve  was4.9Xio~7  g. 
—but  this  is  partly  explained  by  the  fact  that  the 
particular  determination  was  effected  at  25°  C.  Even 
in  this  case  the  decrease  of  carbon  dioxide  was  marked. 
Qualitative  experiments  with  a  2  per  cent  ethyl  urethane 
solution  show  that  even  this  concentration  produces 
a  diminution  of  carbon  dioxide  output. 

CHLORAL  HYDRATE 

As  indicated  in  Table  VIII,  a  2  per  cent  solution  of 
chloral  hydrate  in  sea -water  partially  or  wholly  para- 
lyzes the  nerve  in  ten  minutes  and  recovery  is  appar- 
ently perfect.  A  3  per  cent  concentration  produces 
complete  paralysis  and  the  return  of  excitability  is 
good.  Treatment  with  a  4  per  cent  chloral  hydrate 
solution  for  the  same  period  of  time  also  produces 
paralysis,  but  recovery  is  not  always  good.  In  each 


EXCITATION  AND  CONDUCTION 


case  the  decrease  of  carbon  dioxide  production  is  decided. 
Table  VIII  illustrates  quantitatively  the  difference  in 
carbon  dioxide  production  under  these  different  con- 
ditions. The  interesting  results  with  3  per  cent  and 
4  per  cent  solutions  will  be  considered  later. 

TABLE  VIII 
EFFECTS  OF  CHLORAL  HYDRATE  ON  CLAW  NERVE  OF  SPIDER  CRAB,  Libinia  canaliculata 


TREATED  BY 

EFECTS  ON 
EXCITABILITY 

AFTER 
RETURN  TO 
SEA-WATER 

CHANGE 
IN  WEIGHT 

IN  10 

MINUTES 

AMOUNT  OF  CO* 
PRODUCED  BY 

10  MG.  OF  THE 

NERVE  IN  10 
MINUTES 

Concentra- 
tion in 
Sea-Water 

For 
How 
Long 

o  per  cent  .  .  . 

10  min. 

Excitable 

No  change 

7.9X10—7  g.at2o?2 

o  per  cent  .  .  . 

10  min. 

Inexcitable 

No  change 

5.7X10—7  g.  at  22° 

0.4  per  cent. 

10  min. 

Becomes  more 

Excitable 

No  change 

ii.SXio—7  g.at2o?4 

irritable 

i  per  cent  .  .  . 

10  min. 

Slow  narcosis 

Excitable 

No  change 

Not  determined 

2  per  cent.  .  . 

10  min. 

Partial  or  com- 

Good return 

Very  slight 

4.2X10"  7g.at22?5 

plete  narcosis 

gain 

3  per  cent  .  .  . 

10  min. 

Completely 

Fair  return 

25  per  cent 

2.  SXio-7  g.at23?s 

narcotized 

gain 

4  per  cent  .  .  . 

lomin. 

Completely 
narcotized 

Partial  or 
doubtful 

50  per  cent 
gain 

3.  6Xio~7  g.at23?5 

return 

Is  the  decrease  of  carbon  dioxide  due  to  narcosis? — 
The  results  given  above  establish  beyond  a  doubt  that 
during  treatment  with  narcotics,  in  concentrations  which 
produce  a  reversible  loss  of  irritability,  the  carbon 
dioxide  output  of  a  nerve  is  greatly  reduced.  The 
differences  thus  produced  are  far  beyond  the  limits 
of  experimental  error  and  there  can  be  no  suspicion  that 
the  phenomenon  is  the  result  of  faulty  observation.  The 
question  might  be  raised,  however,  whether  this  diminu- 
tion is  directly  related  to  the  narcosis,  or  whether  it 
might  not  be  due  to  some  factor  casually  introduced 


66  A  CHEMICAL  SIGN  OF  LIFE 

during  the  experiment.  With  this  contingency  in  view, 
certain  possible  objections  are  here  considered. 

The  method  employed  for  carbon  dioxide  determina- 
tion is  so  delicate  that  a  change  in  the  reaction  of  the 
sea-water,  brought  about  possibly  by  the  addition  of 
the  narcotic,  might  be  sufficient  materially  to  alter  the 
values  obtained.  This,  indeed,  is  the  reason  why  we 
have  never  been  able  to  investigate  the  effects  of  potas- 
sium cyanide,  since  the  slight  trace  of  alkalinity  thus 
introduced  seriously  modifies  the  results.  This  objec- 
tion, however,  we  have  been  able  to  refute  by  direct 
experimental  means. 

If  the  solution  of  the  narcotic  differs  in  reaction 
from  sea-water  sufficiently  to  influence  the  determina- 
tion, a  similar  effect  should  be  observed  in  the  case  of  a 
nerve  which  has  been  killed.  Two  freshly  isolated 
nerves  of  approximately  the  same  weight  were  killed 
simultaneously  by  means  of  steam  and  left  for  twenty 
minutes,  one  in  a  2  per  cent  solution  of  chloral  hydrate 
and  the  other  in  sea-water.  A  measurement  of  the 
adventitious  carbon  dioxide  production  from  the  two 
nerves  so  treated  gave  no  evidence  of  any  difference. 
The  diminution  of  carbon  dioxide  from  nerves  subjected 
to  the  action  of  narcotics  cannot,  therefore,  be  referred 
to  any  change  in  the  reaction  of  the  sea-water  produced 
by  the  narcotic. 

Another  possibility  is  involved  in  the  fact  that  certain 
narcotics  produce  phenomena  other  than  those  of  nar- 
cosis. This  is  probably  the  reason  why  the  metabolism 
change  is  never  exactly  the  same  in  the  case  of  two 
nerves  in  which  typical  narcosis  has  been  induced  by 
different  means.  One  of  these  effects  must  be  a  change 


EXCITATION  AND  CONDUCTION  67 

in 'osmotic  pressure,  but  that  the  lowering  of  carbon 
dioxide  output  cannot  result  essentially  from  the  osmotic 
effect  is  evidenced  in  many  indirect  ways.  We  found 
that  the  sciatic  nerve  of  the  frog  when  treated  with  2  per 
cent  ethyl  urethane  solution  gains  about  30  per  cent  in 
weight  during  ten  minutes'  immersion.  No  change  in 
weight,  however,  takes  place  in  the  spider  crab's  nerve 
on  a  similar  treatment  with  the  same  concentration  of 
this  narcotic.  Loss  of  irritability  ensues  in  each  case, 
and  in  each  case  the  carbon  dioxide  production  is  greatly 
diminished.  A  4  per  cent  solution  of  chloral  hydrate 
causes  the  spider  crab's  nerve  to  increase  50  per  cent 
in  weight  in  ten  minutes,  while  4  per  cent  ethyl  urethane 
solution  produces  no  change  of  weight  in  the  same  nerve. 
Yet  both  narcotics  depress  carbon  dioxide  output 
greatly.  That  this  decrease  is  independent  of  osmotic 
effect  is  further  shown  by  our  work  on  the  effect  of  ether 
vapor  on  carbon  dioxide  production  in  a  frog's  nerve. 

In  this,  work  with  a  frog's  nerve  it  was  found  that 
ethyl  urethane  will  reduce  carbon  dioxide  production, 
but  that  soon  after  the  nerve  begins  to  gain  in  weight 
the  tendency  is  for  this  production  to  increase  slightly, 
though  not  sufficiently  to  raise  it  to  its  normal  value. 
Although  this  point  is  still  under  quantitative  investi- 
gation, it  seems  certain  that  this  increase  of  carbon 
dioxide  is  casual,  and  probably  due  to  a  sort  of  water 
rigor.  Somewhat  similar  results  are  obtained  with  the 
spider  crab's  nerve  (Table  VIII).  Thus  we  find  that 
with  a  3  per  cent  chloral  hydrate,  solution  the  carbon 
dioxide  production  is  least — about  one-third  that  of  the 
normal  nerve — while  with  a  4  per  cent  solution  it  is  a 
little  less  than  one-half.  Investigation  of  the  effect  of 


68  A  CHEMICAL  SIGN  OF  LIFE 

this  narcotic  on  the  weight  of  the  tissue  shows  that  a  2 
per  cent  concentration  has  but  little  effect  for  the  first 
ten  minutes,  though  during  the  course  of  half  an  hour  a 
gain  of  50  to  100  per  cent  takes  place.  A  3  per  cent 
concentration  produces  a  gain  of  25  per  cent  in  ten  min- 
utes, while  in  a  4  per  cent  solution  the  nerve  gains  50 
per  cent  in  the  same  space  of  time.  These  results, 
together  with  those  on  the  frog's  nerve,  indicate  that, 
whatever  interpretation  we  put  upon  this  change  in 
weight,  the  result  of  such  a  process  is  temporarily,  at 
least,  to  increase  slightly  the  amount  of  carbon  dioxide 
evolved,  and  that,  so  far  as  the  effects  of  narcotics  are 
concerned  in  our  work,  its  slightly  increased  production 
from  the  narcotized  nerve  which  gains  in  weight  may  be 
looked  upon  as  adventitious.  The  correctness  of  our 
interpretation  of  the  diminution  of  carbon  dioxide  output 
as  an  effect  primarily  connected  with  narcosis  is  further 
supported  by  a  study  of  the  effects  of  weak  concentra- 
tions of  narcotics  for  different  periods  of  time. 

Effects  of  weak  concentrations  of  narcotics  on  carbon 
dioxide  production  in  the  nerve  fiber. — It  is  well  known 
that  the  primary  effect  of  narcotics  is  to  increase  irri- 
tability, after  which  the  typical  depression  follows. 
This  primary  effect  is  well  brought  out  by  the  use  of 
rather  weak  concentrations  of  narcotics.  Although 
we  have  made  no  quantitative  determination  of  the 
degree  of  irritability,  it  is  evident  that  a  nerve  after  ten 
minutes'  immersion  in  a  0.4  per  cent  chloral  hydrate 
solution  has  become  abnormally  irritable.  After  about 
one  hour's  treatment,  however,  the  nerve  finally  becomes 
paralyzed.  If  the  carbon  dioxide  output  of  a  nerve 
treated  for  ten  minutes  with  a  0.4  per  cent  chloral 


EXCITATION  AND  CONDUCTION  69 

hydrate  solution  is  compared  in  the  usual  manner  with 
that  of  a  normal  nerve,  it  is  easily  demonstrated  that  the 
carbon  dioxide  production  of  the  nerve  so  treated  is 
greatly  increased.  The  quantitative  determinations 
tabulated  above  illustrate  this  perhaps  more  con- 
vincingly (see  Table  IX,  horizontal  column  3). 

That  this  change  in  the  carbon  dioxide  production  of 
the  nerve  when  treated  with  the  lower  concentration  is 
closely  connected  with  the  physiological  state  is  further 
demonstrated  in  the  following  experiments.  Many  claw 
nerves  were  isolated  from  several  spider  crabs,  each 
pair  chosen  being  approximately  of  the  same  weight,  and 
of  each  pair  one  was  placed  in  a  o.  4  per  cent  solution  of 
chloral  hydrate  and  the  other  in  sea- water.  A  compari- 
son was  made  of  the  rate  of  carbon  dioxide  production 
of  the  first  pair  in  the  biometer  in  the  usual  manner  at 
the  end  of  ten  minutes;  at  the  end  of  half  an  hour  a 
second  pair  was  compared  similarly,  and  so  on.  The 
result  is  giyen  in  Table  IX.  This  table  is  of  more  than 
passing  interest,  for  it  illustrates  an  easy  source  of  error 
in  the  study  of  narcosis.  Evidently  it  is  of  prime 
importance  to  determine  the  carbon  dioxide  output 
during  a  comparatively  small  time  interval,  rather 
than  during  one  of  long  duration.  If  we  were  to  deter- 
mine the  output  of  the  gas  for  sixty  minutes'  respiration 
of  the  nerve  treated  with  a  o .  4  per  cent  chloral  hydrate 
solution,  we  might  be  led  to  the  conclusion  that  the 
narcotic  has  no  effect  whatever  on  the  metabolic  rate. 
For  although  we  have  shown,  by  taking  corresponding 
nerves  at  the  beginning  and  at  the  end  of  the  narcosis, 
that  the  primary  effect  is  to  increase  carbon  dioxide 
production  and  that  later  it  is  greatly  diminished,  the 


70 


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EXCITATION  AND  CONDUCTION  71 

algebraic  sum  of  these  results  might  readily  approximate 
the  value  found  for  normal  respiration. 

The  general  phenomena  are  the  same  for  ethyl 
urethane,  except  that  the  primary  increase  of  the  gas 
in  light  narcosis  is  much  more  marked,  being  more 
than  twice  as  great  if  compared  with  the  value  for  the 
normal  nerve  at  20?  2  C.  It  was  noted  that  the  nerves 
after  treatment  with  a  i  per  cent  urethane  solution  were 
hyper-irritable,  but  a  part  of  this  large  increase  must 
doubtless  be  due  to  the  higher  temperature  at  which 
the  experiment  was  performed. 

Carbon  dioxide  production  from  "  inexcitable"  normal 
nerve. — During  the  warm  weather  we  occasionally  came 
across  a  claw-nerve  preparation  in  which  no  sort  of  stimu- 
lation of  the  nerve  could  evoke  any  response  whatever, 
although  the  peripheral  organs  were  perfectly  excitable. 
Response  by  the  attached  muscle  to  stimulation  of  the 
claw  nerve  of  the  spider  crab  is  of  three  sorts:  con- 
traction, or  relaxation  of  the  claw,  and  movements  of  the 
lower  joint.  In  general,  different  strengths  of  the 
stimulating  current  result  in  different  responses.  It  was 
at  first  thought  possible  that  in  these  cases  the  stimula- 
tion might  be  calling  forth  opposed  responses,  so  that 
one  neutralized  the  other,  and  thus  that  no  response 
resulted.  But  further  investigation  showed  apparently 
that  the  nerve  was  inexcitable,  since  after  immersion 
in  the  sea -water  irritability  was  often  restored.  To 
whatever  cause  this  may  have  been  due,  the  interesting 
fact  is  that  such  " inexcitable"  nerves  invariably  showed 
an  abnormally  low  rate  of  carbon  dioxide  production. 
The  results  of  the  quantitative  estimates  on  these  nerves 
are  given  in  Tables  VII  and  VIII,  horizontal  column  2. 


72  A  CHEMICAL  SIGN  OF  LIFE 

Summary. — The  main  points  brought  out  by  the 
study  of  narcosis  are:  (i)  carbon  dioxide  production  is 
greatly  diminished  when  the  nerve  is  narcotized  either 
by  chloral  hydrate  or  by  ethyl  urethane  in  concentra- 
tions which  produce  a  reversible  loss  of  excitability; 
(2)  with  a  weak  concentration  of  these  narcotics,  at  the 
beginning,  the  carbon  dioxide  production  is  increased, 
but  later  is  diminished.  This  is  in  accord  with  the  facts 
that  these  concentrations  primarily  stimulate,  or  in- 
crease, the  irritability  of  the  nerve  for  a  time.  The 
conclusion  drawn  from  these  facts  is  that  metabolism  in 
the  nerve  is  interfered  with  by  any  agency  which  inter- 
feres with  the  excitability  of  the  nerve.  Excitability 
and  resting  respiration  go  hand  in  hand. 

The  direction  of  the  nerve  impulse  and  the  metabolic 
gradient.— Although ,  it  has  been  established  that  an 
excitation  wave  travels  in  both  directions  from  the  point 
of  the  stimulus  and  that  this  wave  is  in  all  probability 
identical  with  the  nerve  impulse,  yet  in  the  normal  con- 
dition in  the  body  one  fiber  is  supposed  to  conduct 
the  impulse  in*  one  direction  only.  Based  on  this 
difference  in  the  direction  of  the  conduction,  one  set  of 
the  nerve  trunks  is  called  efferent  and  the  other  afferent, 
according  as  they  conduct  from  or  toward  the  central 
nervous  system.  That  there  is^a  very  interesting  rela- 
tion between  the  direction  in  which  the  impulse  normally 
goes  and  the  rate  of  metabolism  at  different  parts  of  the 
nerve  will  be  set  forth  in  the  following  paragraphs. 

Efferent  fibers. — If  we  take  the  bundle  of  nerves 
between  the  second  and  third  joints  of  the  claw  of  the 
spider  crab  and  cut  it  at  the  middle,  the  two  halves  being 
of  about  equal  weight,  and  place  each  in  a  chamber  of  the 


EXCITATION  AND  CONDUCTION 


73 


biometer  and  measure  its  rate  of  carbon  dioxide  produc- 
tion, we  find  that  the  part  of  the  nerve  nearer  the  body, 
the  proximal  portion,  gives  off  more  gas  than  the  distal 
end  of  the  nerve.  This  nerve  is  mainly  efferent  and 
normally  conducts  the  impulse  down  the  claw  from 
proximal  to  distal  direction.  A  quantitative  estimation 
shows  that  the  proximal  end  gives  more  than  twice  as 
much  carbon  dioxide  as  the  distal,  the  former  giving 
at  least  y.pXio"7  g.  and  the  latter  3.7  io~7  g.  per 
centigram  per  ten  minutes. 

TABLE  X 

CARBON  DIOXIDE  PRODUCTION  FROM  Two  DIFFERENT 
PORTIONS  OF  CLAW  NERVE  OF  SPIDER  CRAB, 
Libinia  canaliculate 


Portion  of  Nerve 

0 

Temperature 
Degrees  C. 

Amount  of  CO. 
Given  OS  by  10  mg. 
of  the  Nerve  in 
10  Minutes 

Whole                      { 

15-16 

6.7Xio~7g. 

Proximal   .  . 

20.2 
21 

7.9X10    7g. 
8.oXio~7g. 

Distal  

23.2 

3-7Xio~7g. 

From  a  study  of  the  various  conditions  which  mod- 
ify this  difference  in  carbon  dioxide  production  in  the 
various  portions  of  the  nerve  we  are  probably  safe  in 
stating  that  we  are  here  dealing  with  a  physiological 
gradient,  experimental  error  playing  no  part.  There 
are  three  physiological  causes  which  might  account  for  a 
different  rate  of  carbon  dioxide  production:  (i)  different 
degrees  of  injury;  (2)  different  rapidity  of  death;  or 
(3)  unequal  rates  of  metabolism.  The  first  alternative 
is  possible  only  on  the  assumption  that  the  proximal 
region  must  be  more  excitable  (greater  susceptibility  to 
an  injury),  which  always  causes  the  production  of 


74  A  CHEMICAL  SIGN  OF  LIFE 

more  carbon  dioxide.  If  we  are  to  assume  'ihe  second 
possibility,  we  must  inquire  why  one  portion  should  die 
earlier  than  the  other.  The  fact  that  an  isolated  nerve 
stays  excitable  for  a  considerable  period  of  time  makes 
this  interpretation  quite  untenable,  although  we  support 
the  idea  set  forth  by  Child  that  the  death  gradient  is 
directly  associated  with  the  metabolic  gradient.  It 
may  be  added  here  that  death  modifies  the  carbon 
dioxide  production  gradient. 

Whatever  interpretation  we  choose,  inasmuch  as  we 
contend  that  the  rate  of  metabolic  activity  as  measured 
by  the  carbon  dioxide  output  is  a  function  of  the  irri- 
tability, we  are  assuming  that  there  must  be  different 
rates  of  metabolism  along  the  normal  nerve  fiber. 
Not  only  that  such  an  assumption  is  correct,  but  also 
that  the  effect  of  injury  and  death  are  only  secondary 
and  minor  factors,  can  be  shown  in  the  light  of  Child's 
experiments  when  the  same  nerve  was  examined,  result- 
ing in  the  confirmation  of  our  results  by  an  entirely 
different  method. 

He  has  shown  that  in  various  concentrations  of  potas- 
sium cyanide,  from  o.ooi  to  o.oi  molecular  in  strength, 
the  fibrillae  of  the  claw  nerve  of  the  spider  crab  after  a 
time  become  irregular  in  outline  and  more  or  less  vari- 
cose, so  that  a  strand  appears  more  or  less  granular  in- 
stead of  fibrillar,  like  a  fresh  nerve.  With  this  criterion 
he  has  discovered  a  gradient  similar  to  our  metabolic 
gradient,  which  appears  in  the  structural  death  changes. 
Using  a  i  per  cent  ethyl  ether  solution  in  sea-water,  or 
even  a  somewhat  lower  concentration,  he  found  that 
the  change  from  fibrillar  to  granular  appearance  begins 
at  the  ends  of  the  nerve  very  soon  after  it  is  brought  into 


EXCITATION  AND  CONDUCTION 


zsz 


the  solution,  but  that  it  does  not  progress  equally  from 
the  two  ends.  A  distinct  gradient  in  this  change  can  be 
seen  extending  peripherally  for  a  few  millimeters  from  the 
central  end  and  a  shorter  distance  centrally  from  the 
peripheral  end.  This  first  change  remains  limited  to 
the  terminal  regions  of  the  nerve  and  is  undoubtedly, 
as  he  interprets  it,  a  temporary  metabolic  gradient  from 
the  ends  inward,  due  to  stimulation  and  injury  resulting 
from  severing  the  nerve  at  these  points.  Later,  however, 
the  fundamental  metabolic  gradient  in  the  nerve  appears, 
in  that  the  change  begins  to  progress  along  the  nerve  from 
the  central  toward  the  peripheral  end ;  but  the  change  at 
the  peripheral  end  progresses  but  slowly,  or  not  at  all, 
in  the  central  direction.  From  this  time  on  a  distinct 
gradient  in  the  change  is  visible  until  it  has  progressed 
along  the  whole  length  of  the  nerve.  Except  in  the  ter- 
minal region  adjoining  the  peripheral  cut  end  the  death 
change  always  progresses  in  the  peripheral  direction. 
The  peripheral  third  of  the  length  may  be  entirely 
unchanged  at  a  time  when  the  central  third  or  more  has 
completely  lost  the  fibrillar  appearance.  Thus  the  fun- 
damental difference  of  the  two  ends  is  made  apparent. 
Child  has  further  shown  that  if  the  nerve  is  crushed 
or  injured  at  any  point  similar  gradients  appear  on  both 
sides  of  the  injury,  but  do  not  extend  to  a  great  distance 
before  the  general  change  reaches  this  region  in  its 
progress  peripherally. 

Since  it  has  been  demonstrated  repeatedly  that 
susceptibility  of  other  tissues  and  organisms  to  reagents 
like  ether  and  cyanide  is  an  expression  of  the  rate  of 
metabolism  in  the  tissue,  these  results  of  Child  not 
only  confirm  our  demonstration  that  there  is  a  clear 

! 


76  A  CHEMICAL  SIGN  OF  LIFE 

centro-peripheral  respiratory  gradient  in  the  claw  nerve 
of  the  spider  crab,  but  also  indicate  very  clearly  that 
this  gradient  exists  in  a  normal  nerve  independent  of 
injury. 

Afferent  fibers. — The  optic  nerve  of  Limulus  was 
tried  next.  It  is  a  non-medullated,  long,  apparently 
uniform,  nerve.  It  can  be  isolated  in  a  length  of  four 
or  five  inches  without  cutting  it  at  either  end,  though 
the  task  is  rather  laborious.  It  is  important  that  the 
peripheral  end  should  be  left  intact  with  the  eyes.  This 
is  accomplished  by  cutting  the  shell  about  two  inches 
square  around  the  eye.  By  gently  lifting  the  eye  with 
the  nerve,  we  can  easily  trace  the  nerve  centrally  up  to 
the  brain  without  any  injury. 

TABLE  XI 

CARBON  DIOXIDE  PRODUCTION  FROM  DIFFERENT  PORTIONS 
OF  OPTIC  NERVE  OF  KING  CRAB,  Limulus 
polyphemus,  FEMALE 


Portion  of  Nerve 

Temperature 
Degrees  C. 

Amount  of  CO. 
Given  Off  by  10  mg. 
of  the  Nerve  in 
10  Minutes 

Whole  

17.8 

2.6Xio~~7  g. 

Proximal  
Distal  

22.5 

22 

3.oXio~~'g. 
5.0X10     7  g. 

When  such  a  long  stretch  of  the  nerve  is  cut  at  both 
ends  simultaneously  and  is  then  divided  at  the  middle 
so  as  to  furnish  two  parts  of  approximately  the  same 
weight,  and  the  rate  of  carbon  dioxide  production  of 
these  two  parts  is  compared,  we  find  that  the  centro- 
peripheral  gradient  discovered  in  the  case  of  the  claw 
nerve  is  exactly  reversed.  In  the  optic  nerve  of  Limulus 
the  proximal  portion  (nearer  to  the  brain  ring)  jives 


EXCITATION  AND  CONDUCTION  77 

much  less  carbon  dioxide  than  the  distal  portion  (nearer 
to  the  retina),  where  the  impulses  normally  originate. 

Table  XI  shows  the  quantitative  results. 

Sensory  dendrites. — These  results  were  at  first 
surprising;  but  they  became  exceedingly  interesting 
when  we  took  into  consideration  the  functional  or 
developmental  difference  between  the  two  nerves. 
The  claw  nerve  of  the  spider  crab  is  believed  to  be 
composed  mainly  of  efferent  fibers,  while  the  optic 
nerve  of  Limulus  is  an  almost  purely  afferent  nerve. 
The  direction  of  the  normal  nerve  impulse  in  one 
of  these  nerves  is,  therefore,  exactly  opposite  to  its 
direction  in  the  other.  .Developmen tally  speaking, 
however,  the  distal  portion  of  the  optic  nerve  corre- 
sponds to  the  proximal  portion  of  the  claw,  nerve  in  that 
these  portions^  are  in  each  case  nearer  the  nerve  cells 
from  which  the  fibers  come.  Thus  our  results  with  the 
two  opposing  gradients  may  be  subject  to  two  alternative 
interpretations.  Either  the  metabolic  gradient  may 
correspond  to  the  developmental  gradient,  i.e.,  all  the 
portion  nearer  to  the  mother-cells  may  have  a  higher  rate 
of  metabolism,  or  it  may  correspond  to  the  functional 
gradient,  i.e.,  the  nearer  the  portion  is  to  the  stimulus 
the  higher  is  the  carbon  dioxide  production. 

This  question  will  be  automatically  solved  if  we 
study  the  metabolic  gradient  of  a  fiber  whose  functional 
direction  is  opposite  to  its  developmental  direction; 
e.g.,  an  afferent  nerve  fiber  lying  peripherally  to  its 
nerve  cells — i:e.ra--sensory  dendrite — should  be  studied. 
Professor  C.  Judson  Herrick  kindly  suggested  that  we 
use  a  lateral  line  nerve,  or  an  accessory  lateral  line 
nerve,  of  a  fish.  In  the  carp  and  the  catfish  both  of 


78  A  CHEMICAL  SIGN  OF  LIFE 

these  nerves  are  present  in  the  body  region,  and  they 
are  wholly  sensory  without  admixture  of  motor  fibers. 
The  fibers  of  both  nerves  are  dendrites  of  ganglion 
cells  which  lie  in  the  head. 

When  these  fibers  were  subjected  to  our  tests  it  was 
found  that  in  every  case  the  proximal  portion  of  the 
fresh  nerve  gave  much  less  carbon  dioxide  than  the  distal 
portion,  indicating  that  the  gradient  is  correlated  with 
function  and  not  with  development.  And  in  this  case 
it  should  be  noted  that,  although  the  fibers  of  both  nerves 
are  dendrites  of  their  respective  neurons  and -conduct 
afferent  impulses,  the  function  of  the  two  nerves  is 
widely  different,  the  lateral  line  nerve  (ramus  lateralis 
vagi)  being  excited  by  water  vibration  and  the  accessory 
lateral  line  nerve  (ramus  lateralis  accessorius)  being 
exclusively  gustatory  in  function.  The  quantitative 
estimates  on  these  nerves  given  in  Table  XII  will  illus- 
trate the  presence  of  this  marked  gradient  of  carbon 
dioxide  of  the  sensory  dendrites. 

TABLE  XII 

CARBON  DIOXIDE  PRODUCTION  FROM  DIFFERENT  PORTIONS 

OF  LATERAL  LINE  NERVE  (RAMUS  LATERALIS 

VAGI)  OF  CARP 


Portions  of 
Nerve 

Temperature 
Degrees  C. 

Amount  of  COa 
Given  Off  by  10  mg. 
of  the  Nerve  in 
10  Minutes 

Proximal  
Distal  

24 
24 

4.9X10—7-  5.2Xio~7g. 
12.4X10—  '-18.5X10—  7g. 

That  the  metabolic  gradient  is  directly  associated 
with  the  functional  direction  is  strikingly  shown  also  by 
further  studies  with  different  fibers  in  which  quantitative 
determinations  were  not  made,  but  in  which  the  output 


EXCITATION  AND  CONDUCTION 


79 


of  carbon  dioxide  of  two  equal  weights  of  proximal  and 
distal  portions  of  the  same  nerve  were  compared  in  the 
bipmeter  under  identical  conditions.  The  portion  of 
the  nerve  which  gave  off  sufficient  carbon  dioxide  to 
produce  the  precipitate  of  barium  carbonate  in  the 
shortest  time  and  the  greatest  abundance  evidently 

produced  the  most  carbon  dioxide. 

. 

TABLE  XIII 

COMPARATIVE  STUDIES  ON  RATE  OF  CARBON  DIOXIDE  PRODUCTION  FROM  Two 
PORTIONS  OF  VARIOUS  NERVES 


TEMPERA- 

"\TFB\TI 

TURE  OF 

JMERV. 

B 

AMOUNTS 

DATE 

ROOM 

OF  COa 

DEGREES 

COMPARED 

C. 

Name  of 

Kind  of 

Portion  of 

Weight  of 

Aug.  31... 

23.2 

Optic  nerve  of 
Limulus 

Afferent 
mainly 

Proximal 
Distal 

17      mg. 
17 

Less 
More 

Sept.  15... 

18 

Optic  nerve  of 

Proximal 

23 

Less 

skate 

u 

Distal 

23 

More 

Feb.  15  ... 

22 

R.  lat.  vagi  of 

Proximal 

4-4 

Less 

carp 

a 

Distal 

More 

Dec.  20  ... 

2O 

R.  lat.  vagi  of 

Proximal 

Less 

catfish 

« 

Distal 

.8 

More 

Dec.  13  ... 

17 

R.  lat.  ace.  of 

Proximal 

•  4 

Less 

catfish 

a 

Distal 

•4 

More 

Nov.  23  ... 

25.5 

Posterior  root 

Proximal 

•  5 

Less 

of  dog 

a 

Distal 

6 

More 

Aug.  30.  .. 

21.8 

Claw  nerve  of 

Efferent 

Proximal 

32 

More 

Feb.  26... 

IQ 

spider  crab 
Hypoglossal 

mainly 

Distal 
Proximal 

44 
Equal 

Less 
More 

Feb.  21... 

23 

nerve  of  dog 
Same  nerve  of 

" 

Distal 
Proximal 

weights 
Equal 

Less 

More 

carp 

• 

Distal 

weights 

Less 

Sept.  15... 

17-8 

Oculomotor 

Proximal 

ii 

More 

nerve  of  skate 

• 

Distal 

ii 

Less 

Nov.  26.  .. 

26 

Anterior  root 

Proximal 

,      1.6 

More 

of  dog 

Distal 

1.8 

Less 

While  we  have  not  by  any  means,  exhausted  the 
various  kinds  of  nerve  fibers  available  for  the  study 
of  this  gradient,  the  results  obtained  in  those  nerves 
already  examined  have  been  very  uniform  and  the 
nerves  themselves  have  had  varied  functions  and  have 
come  from  several  different  classes  of  animals,  namely, 


8o     •  A  CHEMICAL  SIGN  OF  LIFE 

fishes,  mammals,  crabs,  and  arachnids.  We  feel  certain, 
therefore,  that  the  existence  of  a  metabolic  gradient 
in  nerve  fibers  correlated  with  the  functional  activity,  and 
related  to  the  direction  the  nerve  impulse  takes,  must  be 
a  very  general  phenomenon.  From  these  facts  we  come 
to  the  very  simple  conclusion  that  is  summarized  in  the 
following  statement:  The  normal  nerve  impulse  passes 
from  a  point  of  higher  toward  a  point  of  lower  carbon 
dioxide  production — from  the  more  irritable  to  the  less 
irritable  parts.  There  is  also  a  decrement  in  the  nerve 
impulse.  It  cannot  proceed  indefinitely  along  a  nerve; 
it  will  ultimately  die  out. 

Velocity  of  the  nerve  impulse  and  its  relation  to  respira- 
tion.— If  the  nerve  impulse  cannot  pass  through  the 
fiber  without  consuming  substance,  it  is  reasonable  to 
expect  that  there  may  be  some  relation  between  the 
rate  of  production  of  carbon  dioxide  in  the  resting  nerve 
and  the  velocity  of  the  nerve  impulse.  The  reason  for 
such  a  supposition  is  clear.  It  has  already  been  indi- 
cated that  the  more  irritable  a  nerve  is  the  more  carbon 
dioxide  does  it  produce  in  the  resting  state.  Hence, 
the  more  it  respires,  the  more  irritable  it  is,  the  faster 
should  it  conduct  the  impulse.  This  is  actually  found, 
within  limits,  to  be  the  case.  If  one  compares  corre- 
sponding nerves  of  different  animals,  or  different  nerves 
of  the  same  animal,  it  is  found,  other  things  being  equal, 
that  there  is  a  relation  between  the  speed  of  contraction 
of  the  muscles  supplied  by  the  nerve  and  the  velocity 
with  which  the  nerve  supplying  the  muscle  conducts 
the  impulse.  Obviously  it  would  be  foolish  to  have 
a  very  rapidly  contracting  muscle,  or  limb,  supplied  with 
a  nerve  which  conducted  the  impulse  to  the  muscle  at  a 


EXCITATION  AND  CONDUCTION  81 

vety  slow  rate.  And,  being  foolish,  this  does  not  happen. 
We  can  use  the  speed  of  contraction,  therefore,  as  a  rough 
index  of  the  relative  velocities  of  the  nerve  impulses  in 
nerves  supplying  the  muscles.  In  this  way  the  rates  of  the 
nerve  impulse  of  the  ambulacral  nerves  of  the  king  crab, 
the  spider  crab,  and  the  lobster  are  in  the  ratio  of  1:2:4. 
This  ratio,  as  far  as  the  king  crab  and  the  spider  crab  are 
concerned,  is  very  nearly  the  same  as  that  found  for  the 
carbon  dioxide  output  of  these  nerves,  as  may  be  seen 
in  Table  IV  (p.  32).  Low  respiration,  low  irritability, 
and  low  speed  of  conduction  appear  to  go  together. 

We  can  also  test  the  rate  of  metabolism  in  these 
nerves  indirectly  by  Child's  method,  and  this  gives  us 
just  the  same  result.  This  method  consists  in  determin- 
ing the  speed  with  which  the  excitability  of  the  nerves 
is  abolished  when  they  are  treated  with  the  same  con- 
centration of  a  narcotic.  The  greater  the  rate  of  metabo-. 
lism  the  more  susceptible  is  the  tissue  to  narcotics.  We 
found  in  the  case  of  these  three  nerves  that  the  excita- 
bility of  the  lobster  nerve  is  most  quickly  abolished, 
then  that  of  the  spider  crab,  and  finally  that  of  the  king 
crab.  This  is  the  same  order  in  which  the  nerves  con- 
duct the  impulse,  the  lobster  conducting  fastest.  This 
is  therefore  additional  proof  of  the  fact  that  the  speed 
of  the  impulse  and  the  degree  of  metabolism  in  the 
resting  nerve  are  correlated,  at  least  in  these  nerves.  It 
is  remarkable  to  note  that  the  carbon  dioxide  output 
of  the  nerves  of  Limulns,  the  king  crab,  is  very  low  in 
comparison  with  that  of  other  animals.  This  may  be 
correlated  with  the  very  sluggish  behavior  of  this  animal 
and  its  power  of  living  for  a  long  time  without  food  and 
with  very  little  air. 


82  A  CHEMICAL  SIGN  OF  LIFE 

While  there  seems,  then,  to  exist  a  very  close  corre- 
lation between  the  rate  of  respiration  of  resting  nerves 
and  the  velocity  with  which  they  conduct  a  nerve 
impulse,  the  data  for  the  establishment  of  this  generaliza- 
tion must  necessarily  be  cumulative,  and  we  are  not  yet 
able  to  state  positively  that  all  nerves  which  give  off 
much  carbon  dioxide  in  the  resting  state  will  be  found 
to  conduct  the  impulse  more  rapidly  than  those  which 
give  off  less.  There  are,  however,  several  conditions 
which  influence  the  rate  of  the  nerve  impulse,  and  we 
have  investigated  the  effect  of  these  conditions  on  the 
production  of  carbon  dioxide.  Two  of  these  con- 
ditions are:  changes  in  the  salts  in  the  nerves,  and 
temperature. 

a)  Changes  in  salts:  Mayer  found  that  the  rate  of 
nervous  conduction  in  the  sub-umbrella  regions  of  the 
subtropical  jelly  fish,  Medusa  cassiopea,  increases  about 
5  per  cent  in  sea-water  diluted  with  distilled  water  in  the 
proportion  of  9:1,  while  it  decreases  50  per  cent  in  sea- 
water  diluted  to  50  per  cent  with  fresh  water.  By  sub- 
stituting 0.9  M  dextrose  for  the  distilled  water  he 
demonstrated  that  the  change  in  rate  of  the  impulse  in 
diluted  sea-water  was  not  due  to  the  decrease  of  osmotic 
pressure,  but  was  due  to  the  change  in  concentration  of 
the  salt.  If  under  those  conditions  which  decrease 
the  rate  of  the  conduction  a  measurement  is  made  of  the 
amount  of  carbon  dioxide  produced  from  the  thin  layer 
of  the  regenerating  ectoderm  tissue,  it  is  found  that  a 
change  in  the  rate  of  carbon  dioxide  production  goes 
parallel  with  the  decrease  in  the  rate  of  conduction.  As 
a  result  of  using  the  regenerating  tissue  just  mentioned, 
the  nervous  tissue  regenerates  before  the  muscular,  so 


EXCITATION  AND  CONDUCTION  83 

that  we  can  in  this  way  eliminate  the  effect  of  the  salt  on 
the  muscles. 

b)  Temperature:  It  is  well  known  that  a  change  in 
temperature  affects  the  speed  of  the  nerve  impulse;  an 
increase  of  10°  C.  increases  the  velocity  of  the  impulse  by 
four-fifths,  or  even  more.  This  is  very  significant  in 
view  of  the  fact  that  for  most  physical  processes  the  same 
increase  in  temperature  increases  the  velocity  of  the 
process  by  at  most  one-fifth.  Chemical  processes  are 
accelerated  about  100  per  cent.  While  the  magnitude 
and  variation  of  the  temperature  coefficient  of  velocity 
of  a  physiological  process  do  not  necessarily  tell  us  what 
kind  of  reaction  is  involved  in  the  process,  they  never- 
theless indicate  in  this  instance  very  clearly  that  con- 
duction by  a  nerve  is  not  a  purely  physical  process,  as 
some  have  imagined  it.  It  is  very  important,  evi- 
dently, that  we  should  compare  the  effect  of  temperature 
on  the  carbon  dioxide  output  with  its  effect  on  speed 
of  conduction. 

We  have  made  studies  of  the  metabolic  rate  of  the 
nerve  of  the  king  crab  at  different  temperatures,  such  as 
naturally  occur  at  Woods  Hole  and  at  Dry  Tortugas,  and 
we  have  discovered  that  the  temperature  coefficient  of  the 
production  of  carbon  dioxide  by  the  resting  nerve  is 
just  about  the  same  as  the  temperature  coefficient  of 
the  speed  of  conduction.  A  similar  result  was  obtained 
with  a  sciatic  nerve  of  a  frog  under  experimental  changes 
of  temperature.  We  thus  have  this  additional  point  of 
parallelism  between  the  rate  of  conduction  and  the  pro- 
duction of  carbon  dioxide  in  the  resting  nerve. 

It  is  extremely  interesting  and  significant  that  the 
fundamental  condition  for  the  conduction  of  a  nerve 


84  A  CHEMICAL  SIGN  OF  LIFE 

impulse  is  determined  by  the  chemical  change  going  on 
in  the  nerve  at  the  time  of  stimulation,  and  that  it  is 
the  resting  respiration,  or  metabolism,  which  seems  to 
determine  how  fast  the  nerve  impulse  should  travel  along 
the  fiber.  It  is  exactly  as  if,  during  rest,  the  nerve  sub- 
stance was  sustained  in  a  very  unstable  state  by  the 
expenditure  of  energy  by  processes  which  set  free  the 
carbon  dioxide.  It  is  as  if  the  irritable  or  unstable  con- 
dition was  like  a  stone  rolled  partly  up  a  hill  and  kept 
there  at  the  cost  of  considerable  panting  by  the  toiling 
demon  of  life.  When  the  nerve  impulse  comes  along, 
the  stone  escapes  from  his  grasp  and  rolls  downhill. 
During  the  period  of  rest  or  recovery  which  follows, 
this  tiny,  toiling  Sisyphus,  with  infinite  labor  and  pant- 
ing, pushes  the  stone  uphill.  The  higher  he  gets  it  the 
more  he  gasps,  the  more  unstable  it  becomes  the  more 
easily  it  escapes  his  grasp,  the  more  rapidly  does  it  crash 
down,  and  the  more  irritable  is  the  nerve  the  more 
rapidly  does  the  impulse  travel. 

Conclusion. — Basing  our  conclusions  on  the  foregoing 
experimental  facts,  we  may  express  the  relation  between 
excitation,  conduction,  "and  respiration  in  nerves  as 
follows : 

The  maintenance  of  chemical  activity,  or  metabolism, 
is  responsible  for  that  unstable  condition  in  the  nerve, 
whatever  its  nature,  which  we  call  the  state  of  irrita- 
bility or  excitability.  All  irritable  tissue  must  respire. 
The  tissue  cannot  be  made  irritable  and  then  kept  so 
without  effort.  Chemical  energy  must  constantly  be 
expended  to  keep  the  tissue  irritable.  The  amount  of 
this  expenditure  of  energy  is  not  the  same  at  all  points 
along  the  fiber,  but  it  diminishes  in  one  direction  or  the 


EXCITATION  AND  CONDUCTION  85 

other,  generally   toward    the   central   nervous   system 
in  sensory  nerves  and  toward  the  periphery  in  motor      S* 
nerves.  «^Trie  nerve  impulse,-  generally  travels  in  thd 
direction  of  this  gradient.     When  we  stimulate  a  nerve 
at  any  point,  the  stimulation  consists  in  the  local  in- 
crease of  metabolic  activity  at  the  point  of  irritation. 
The  irritability  is  raised  and  the  carbon  dioxide  output      / 
is  increased  at  that  point  above  the  production  on  either     (  / 
side  of  it.     This  causes  a  local  metabolic  gradient  in     J 
the  nerve  in  both  directions  from  the  point  of  excitation, 
but  the  difference  between  this  point  and  its  surroundings 
will  be  greater  on  one  side  than  on  the  other,  owing 
to  the  gradient  in  the  nerve  just  mentioned.     If  this 
state   of  excitation  is  sufficiently  great,  it  upsets  the 
equilibrium  and  the  impulse  will  be  propagated  in  each 
direction   from    the   point   of   excitation.     The   possi-     !   r\ 
bility  exists  that  it  ought  to  travel  more  easily  in  the 
direction  which  the  nerve  impulse  normally  takes,  and  it 
ought  to  be  possible,  with  a  proper  amount  of  stimulus, 
to  start  a  propagation  in  only  one  direction  from  the 
point  of  stimulus,  but  we  have  not  yet  tested  this  possi- 
bility  experimentally.     The   excitation   always   travels    , 
from  the  point  where  the  excitation  is  greatest  to  that  l 
where  it  is  less.     The  repair  process,  or  the  anabolic 
process,  is  also  propagated. 

The  conditions  which  affect  the  rate  of  nervous 
metabolism  not  only  alter  the  state  of  excitability  of  the 
nerve,  but  also  change  the  speed  of  the  conduction  of  the 
state  of  that  excitation.  Although  we  have  no  evi- 
dence to  show  that  the  chemical  change  itself  constitutes 
the  nerve  impulse,  the  conclusion  is  almost  inevitable 
that  the  nerve  impulse  is  brought  about  by,  or  is  itself 


86  A  CHEMICAL  SIGN  OF  LIFE 

nothing  else  than,  a  propagation  of  chemical  changes, 
the  propagation  in  wave  form  being  due  to  the  restora- 
tion of  an  unstable  equilibrium  disturbed  by  the  increase 
of  metabolism  at  the  point  of  stimulus.  This  prop- 
'agation  is  always  toward  the  point  where  there  is  less 
chemical  activity,  as  measured  by  the  carbon  dioxide 
output. 


CHAPTER  V 
CHEMICAL  SIGNS  OF  LIFE 

We  have  endeavored  to  show  that  the  living  nerve, 
as  long  as  it  is  irritable,  is  chemically  active  and  that 
when  it  functions  this  metabolism  is  accelerated.  As  the 
irritability  of  the  nerve  varies,  there  are  simultaneous 
changes  in  chemical  activity.  What  characterizes  the 
living  state  is  respiration  and  its  increase  on  stimulation. 

We  have  come  now  to  our  main  inquiry,  namely, 
whether  or  not  all  living  matter  undergoes  respiration 
as  long  as  it  is  alive,  and  whether  stimulation  always 
increases  its  respiration.  In  addition,  we  have  to  ask 
whether,  if  this  is  true,  it  can  be  used  as  a  sign  of  life  in 
all  living  matter. 

Seeds. — It  has  hitherto  been  maintained  that  since 
dry  seeds  do  not  respire  but  are  irritable,  irritability  is 
independent  of  respiration.  The  work  of  Horace  Brown, 
Thistleton  Dyer,  and  others  indicates  that  dry  seed  can 
be  kept  alive  at  very  low  temperatures  in  conditions 
where  no  ordinary  gaseous  exchange  is  possible.  It  is 
argued,  therefore,  that  life  is  possible  without  any 
metabolic  activity.  Dry  seeds,  kept  for  long  periods 
in  a  closed  vessel,  have  not  been  found  to  give  any  evi- 
dence of  this  fundamental  chemical  change  occurring 
in  living  matter,  namely,  the  production  of  carbon 
dioxide.  Such  seeds,  it  is  well  known,  are  not  really 
dead,  for  under  proper  conditions  they  germinate. 
They  appear  to  live  without  respiration,  but  this  is  but 

87 


A  CHEMICAL  SIGN  OF  LIFE 

an  appearance.  The  seeds  really  respire.  Waller  con- 
sidered that  our  present  chemical  technique  is  not  refined 
enough  to  reveal  to  us  the  smallest  and  most  infinitesimal 
chemical  changes  which  may  be  going  on  in  the  appar- 
ently dry  and  perfectly  dormant  seed.  He  based  his 
hypothesis  on  two  considerations:  First,  was  the  fact 
that  the  seeds  wear  out,  as  shown  by  their  losing  their 
power  of  germination  and  growth  in  proportion  to  the 
length  of  time  they  have  been  kept.  The  deterioration 
is  more  or  less  rapid,  according  to  the  nature  of  the 
seed  and  the  character  of  the  protective  coats,  but  in 
every  instance  there  is  deterioration  sooner  or  later. 
He  attributes  this  gradual  deterioration  to  chemical 
activity  in  the  seed. 

In  the  second  place,  there  was  the  fact,  which  he 
showed  by  his  electrical  method,  that  a  living  seed  not 
only  differs  from  the  dead  one  in  respect  to  its  electrical 
response,  but  that  the  amount  of  its  response  varies 
according  to  its  age.  Thus,  if  he  took  a  living  seed,  a 
dead  seed  killed  by  heat,  and  a  very  old  Egyptian  seed 
from  about  the  Twelfth  Dynasty  (about  4,400  years 
old)  and  determined  their  electrical  response,  he  found 
a  very  interesting  result.  The  first,  or  living,  seed  gave 
a  large  electromotive  force,  while  the  others,  the  old 
as  well  as  the  dead,  gave  none.  If  he  took  a  group  of 
seeds  from  crops  of  different  years,  he  found  that  there 
was  also  a  gradual  decline  in  the  electrical  response  as 
the  seed  became  old.  He  considered  this  electrical  sign 
as  the  expression  of  the  chemical  changes  which  cannot 
otherwise  be  determined,  and  such  a  sign  of  death, 
according  to  him,  is  manifested  long  before  microscopic 
or  chemical  changes  can  be  detected. 


CHEMICAL  SIGNS  OF  LIFE  89 

1  He  found  that  this  electrical  change — the  blaze 
current,  as  he  called  it — which  appeared  when  the  living 
seed  was  stimulated  by  a  strong  induction  current  was 
not  confined  to  seeds,  but  occurred  also  in  other  varieties 
of  living  matter,  such  as  the  eyeball,  skin,  leaves,  petals, 
and  many  other  tissues  of  plants  and  of  animals.  This 
momentary  electrical  change  produced  thus  only  by 
living  matter  is  accordingly  a  reliable  sign  of  life,  since 
it  does  not  occur  in  dead  matter. 

When  we  discovered  that  even  a  resting  nerve  gave 
off  carbon  dioxide  if  we  used  a  sensitive  method,  we 
at  once  proceeded  with  some  curiosity  to  determine 
whether  or  not  ordinary  seed  is  chemically  inactive. 
We  had  in  mind  thus  to  test  Waller's  conclusion  that  the 
electrical  sign  in  the  seed  is  really  the  sign  of  chemical 
changes  which,  however,  were  not  large  enough  to  be 
detected  by  ordinary  chemical  methods. 

Resting  metabolism  in  seeds. — If  a  few  kernels  of 
wheat  are  placed  in  one  chamber  of  the  biometer,  there 
is  no  difficulty  in  showing  that  seeds  give  off  carbon 
dioxide,  since  a  drop  of  barium  hydroxide  in  the  chamber 
containing  the  seeds  becomes  covered  after  a  time  with 
a  precipitate  of  barium  carbonate.  It  is  true  that  the 
amount  of  carbon  dioxide  given  off  is  exceedingly  small, 
being  many  times  less  than  that  of  the  resting  nerve, 
but  that  this  carbon  dioxide  is  produced  by  a  vital 
metabolism  is  shown  by  the  fact  that  living  seeds 
give  far  larger  amounts  of  the  gas  than  dead  ones. 
A  seed  respires,  therefore,  as  long  as  it  is  alive;  and 
we  can  measure  the  amount  of  respiration.  Of  course 
the  mere  production  of  this  gas  from  a  seed  does  not 
mean  necessarily  that  the  seed  is  alive,  for  the  reason 


90  A  CHEMICAL  SIGN  OF  LIFE 

(already  discussed  on  p.  34)  that  carbon  dioxide  is 
produced  by  many  purely  chemical  processes.  So  we 
proceed  to  inquire  whether  the  output  is  increased  on 
stimulation. 

Increased  metabolism  in  seeds. — The  most  interesting 
thing  ascertained  was  that  the  living  seed,  like  any  other 
living  tissue,  can  be  made  to  give  off  more  carbon  dioxide 
on  stimulation.  It  responds  to  an  injury  and  is,  there- 
fore, irritable.  It  has  already  been  stated  that  a  nerve 
injured  by  crushing  gives  off  more  carbon  dioxide  than 
a  fasting  nerve,  just  as  if  it  had  been  stimulated  by  an 
electrical  shock.  Since  there  was  no  way  of  telling 
what  strength  of  electrical  stimulation  was  required 
in  order  to  arouse  the  seed,  we  stimulated  it  by  an 
injury,  namely,  by  crushing  it.  The  seed  thus  stimu- 
lated showed  a  marked  acceleration  of  its  respiration. 
If  two  apparently  living  kernels  of  wheat  are  taken  and 
one  of  them  is  crushed  and  their  carbon  dioxide  produc- 
tion is  compared  in  the  biometer,  the  crushed  one  always 
produces  more  carbon  dioxide  than  the  normal  one. 
That  this  is  a  vital  response  is  shown  by  the  fact  that  only 
living  seeds  behave  in  this  way.  If  one  takes  two  kernels 
of  any  similar  seed,  which  have  been  killed  in  an  elec- 
trical oven  heated  to  60°  C.,  and  one  of  them  is  crushed, 
there  is  no  difference  in  the  carbon  dioxide  output  of  the 
two  seeds.  The  difference  in  amount  of  carbon  dioxide 
produced  by  crushing  cannot  be  observed  in  dead  seeds 
or  in  anesthetized  seeds.  In  this  respect  a  seed  and  a 
nerve  are  alike;  the  chemical  signs  of  irritability  are 
identical.  Both,  as  long  as  they  are  alive,  respond  to  a 
mechanical  stimulation  by  producing  more  carbon 
dioxide. 


CHEMICAL  SIGNS  OF  LIFE  91 

Is  an  injury  a  stimulation? — Are  we  justified  in 
regarding  the  increase  of  carbon  dioxide  following  injury 
by  crushing  as  in  the  same  category  as  the  increase  of 
carbon  dioxide  production  by  ordinary  stimulation? 
That  this  conclusion  is  justified  is  shown  by  the  fact 
that  such  an  acceleration  of  carbon  dioxide  production 
will  not  take  place  in  inexcitable  tissue.  Neither  killed 
nor  narcotized  tissue  can  be  made  to  give  off  more  carbon 
dioxide  when  crushed.  Response  to  an  injury  is  given 
by  living  irritable  tissue  only.  It  is  impossible  to  injure 
the  dead  tissue. 

Other  tissues. — When  we  discovered  that  the  irrita- 
bility of  a  kernel  of  wheat  and  that  of  the  nerve  fiber  are 
identical,  so  far  as  their  metabolic  expressions  are  con- 
cerned— i.e.,  no  irritability  without  resting  metabolism, 
increased  metabolism  on  stimulation,  and  changes  in 
metabolic  condition,  according  to  the  state  of  excita- 
bility— we  thought  it  might  be  possible  that  this  similar- 
ity between  the  nerve  and  wheat  is  special,  and  that 
other  plant  tissues  may  not  behave  at  all  in  the  same 
way  as  do  seeds.  Similar  experiments  were  consequently 
tried  on  several  other  seeds,  including  wild  oats,  Lincoln 
oats,  Swedish  select  oats,  rice,  corn,  mustard,  and  various 
others,  with  the  result  that,  although  the  amounts  of 
carbon  dioxide  given  off  varied  considerably,  all  living 
seeds  were  found  to  be  metabolically  active.  All  of 
them  responded  to  an  injury,  giving  off  more  carbon 
dioxide  on  crushing.  And  in  no  case  did  we  succeed  in 
producing  more  carbon  dioxide  on  crushing  killed  seeds, 
or  seeds  which  had  lost  germinating  power.  Thus  we 
made  certain  that  under  the  experimental  conditions  in 
the  biometer  it  is  possible  to  detect  the  fundamental 


92  A  CHEMICAL  SIGN  OF  LIFE 

difference  between  dead  and  living  seeds,  namely,  the 
matter  of  metabolic  acceleration  on  injury.  The  in- 
creased carbon  dioxide  production  on  stimulation  is  the 
chemical  sign  of  life  of  seeds  and  tissues  generally,  as 
well  as  of  nerves. 

Once  this  interesting  similarity  between  seeds  and 
nerves  was  well  established  we  made  further  investi- 
gations on  other  plant  tissues,  in  which  conditions 
were  somewhat  different.  It  was  possible  that  the 
removal  of  the  heavy  coat  from  seeds  in  crushing  them 
might  have  something  to  do  with  the  increased  metabolic 
activity,  and  that  this  activity,  therefore,  might  not  be 
manifested  by  all  tissues.  In  fact,  Crocker  has  shown 
that  the  removal  of  the  coat  is  one  of  the  factors  which 
initiates  germination  in  dormant  seeds.  When  we  tried 
different  leaves,  however,  they  all  behaved  in  the  same 
manner  as  did  seeds  and  nerves.  The  leaves  selected 
for  test  were  necessarily  small,  with  the  object  in  view 
of  being  able  to  place  the  whole  leaves  in  the  chamber 
with  the  least  injury.  They  included  such  as  Japanese 
ivy,  common  grass,  Australian  pine,  and  various  others. 
We  may  add  here  that  the  increase  of  carbon  dioxide 
output  as  a  result  of  some  other  forms  of  injury  in  leaves 
has  been  recorded  by  several  investigators. 

Some  objection  might  be  made  against  our  experi- 
ments, however,  on  the  ground  that  the  injury  to  the 
stomata  may  be  responsible  for  the  output  of  more  gas 
in  the  case  of  leaves.  That  this  is  not  the  sole  cause 
of  the  escape  of  the  gas  is  shown  by  our  experiments  on  a 
plant  tissue  without  stomata.  Red  algae  were  tested 
at  the  suggestion  of  Professor  Osterhout  and  gave 
similar  responses. 


CHEMICAL  SIGNS  OF  LIFE  93 

s  Thus  we  extended  our  experiments  to  the  best-known 
tissues  in  the  plant  and  animal  kingdoms,  and  found 
no  exception  to  the  general  rule  cited.  These  results 
surely  justify  the  generalization  that  all  living  tissues 
differ  from  all  dead  tissues  in  that  they  respond  to 
injury,  producing  more  carbon  dioxide  than  the  normal 
tissues;  and  that  by  measuring  this  output  of  the  gas 
in  comparison  with  the  uninjured  we  can  detect  the 
vitality  of  the  tissue. 

Chemical  sign  of  life. — We  have  now  come  to  a  con- 
clusion on  all  the  facts  that  we  have  presented  so  far. 
Of  all  the  signs  of  living  processes  irritability  is  one  of 
the  most  universal.  This  phenomenon  of  irritability  is 
expressed  in  the  power  of  feeling  the  external  world.  It 
is  the  inherent  power  of  the  living  to  react  against  a 
stimulation.  The  necessary  condition  for  this  irritability 
of  tissues  is  metabolic  activity.  Although  this  chemical 
condition  is  necessary  for  all  tissue  in  order  that  it  shall 
be  irritable,  yet  it  is  not  a  sufficient  criterion  for  the 
detection  of  vitality  in  it.  We  must  inaugurate  a  further 
test  of  whether  or  not  it  reacts  chemically  to  a  stimula- 
tion. In  order  to  test  this  power,  we  injure  the  tissue 
and  watch  the  response.  If  the  tissue  is  alive,  me- 
chanical crushing  will  produce  a  metabolic  response; 
if  it  is  not  alive,  there  is  no  response. 

The  detail  of  testing  the  vitality  of  a  tissue  is  as 
follows : 

In  order  to  test  that  of  a  seed,  take  two  or  more 
kernels  of  the  seed  in  question  having  about  equal 
weights.  One  is  placed  in  the  right  chamber  of  the 
biometer,  and  the  other  is  crushed,  or  is  cut  to  pieces, 
and  placed  in  the  left.  The  apparatus  is  filled  with 


94  A  CHEMICAL  SIGN  OF  LIFE 

air  free  from  carbon  dioxide,  and  a  drop  of  barium 
hydroxide  is  introduced  at  the  top  of  the  tube  in  each 
chamber.  If  the  crushed  seed  gives  off  more  carbon 
dioxide  than  the  uncrushed,  as  evidenced  by  a  larger 
deposit  of  the  carbonate  on  the  top  of  the  drop  in  the 
chamber  containing  the  crushed  seed,  the  seed  is  alive. 
If  the  seeds  are  alive,  such  a  distinction  in  the  carbon 
dioxide  output  will  be  noticeable  in  a  few  minutes  in 
some  cases,  or  in  an  hour  or  more  in  other  cases,  all 
depending  on  the  size,  the  number,  and  the  kind  of  seed 
we  are  testing.  With  several  seeds,  as  with  the  fresh 
nerve  of  a  frog,  we  can  detect  vitality  in  this  way  in  a 
few  minutes. 


CHAPTER  VI 
CONCLUSIONS 

Summary. — While  we  cannot  define  life  in  a  physical 
sense,  for  the  reason  that  we  have  no  measure  of  the 
psychic  phenomena  shown  by  living  things,  and  these 
psychic  phenomena  are,  after  all,  the  most  important 
of  the  characteristics  of  life,  there  are  nevertheless  certain 
phenomena  associated  ahyays  with  the  living  processes 
which  are  so  characteristic  that  for  the  majority  of 
organisms  with  which  we  are  familiar  we  have  no  diffi- 
culty in  determining  whether  they  are  living  or  dead. 
Irritability  is  the  universal  sign  of  life,  and  by  it  living 
matter  adjusts  itself  to  its  environment.  The  sign 
of  this  irritability  is  the  functional  power  of  the  tissues. 
Thus  by  measuring  the  functional  power  we  can  speak 
of  measuring  the  amount  of  irritability.  The  changes 
of  a  physical  or  chemical  kind  which  accompany  this 
functioning  are  very  important  for  an  understanding  of 
the  living  process,  for  when  we  know  them  completely  we 
shall  probably  understand  the  nature  of  irritability  itself. 

In  chapter  ii  we  showed  how  it  happened  that,  because 
of  the  apparent  exception  in  the  case  of  nerves,  it  has 
been  generally  concluded  that  chemical  changes  could 
not  be  considered  to  be  essential  to  all  living  processes. 
Some  of  these  changes,  it  appeared,  must  be  due  solely 
to  physical  processes,  and  for  this  reason  irritability 
had  come  to  be  regarded  as  a  purely  physical  phenome- 
non. Various  hypotheses  had  been  made  to  explain  how 

95 


96  A  CHEMICAL  SIGN  OF  LIFE 

this  could  be;  the  change  in  state,  which  was  the  essence 
of  irritability,  was  pictured  as  a  change  in  the  state  of 
the  colloids  or  the  structure  of  the  protoplasm,  or,  more 
recently,  in  the  state  of  its  permeability.  But  how  on 
this  basis  irritability  was  to  be  understood  was  by  no 
means  clear.  On  examining  the  irritability  of  nerves— 
the  apparent  exception  which  had  led  to  the  conclusion 
that  irritability  had  a  physical  and  not  a  chemical 
basis — we  found  that  this  apparent  exception  was 
really  due  to  the  fact  that  our  methods  had  not  hitherto 
been  sufficiently  delicate  to  detect  the  chemical  changes 
which  accompanied  the  process.  By  devising  a  new 
method  for  the  study  of  carbon  dioxide — one  of  the 
terminal  products  of  metabolism  everywhere — we  found 
that  living  nerve  fibers  in  reality  were  undergoing 
chemical  change  at  quite  a  remarkable  rate  and  were  pro- 
ducing carbon  dioxide  faster  than  any  other  tissue  of  the 
body,  if  equal  weights  were  compared.  And  we  found, 
further,  that  reagents  or  physical  methods  which  change 
the  state  of  excitability  of  the  nerve  changed  also  the 
rate  at  which  it  was  producing  carbon  dioxide,  so  that 
the  gas  production  was  evidently  correlated  with  its 
vitality  and  not  with  adventitious  processes. 

In  chapter  iii  we  found  that  although  the  chemical 
activity  is  a  necessary  condition  for  all  living  nerves,  yet 
by  itself  it  is  not  a  demonstrative  sign  of  life;  i.e.,  it  is  not 
a  sufficient  criterion  of  living.  An  additional  criterion  is 
needed  in  order  to  be  sure  that  any  tissue  is  living. 
In  the  case  of  the  nerve,  we  demonstrated  that  this 
additional  sign  was  also  present.  This  sign  is  the  fact, 
that  all  living  matter,  including  the  nerve,  responds  to 
a  stimulus  by  the  production  of  more  carbon  dioxide.' 


CONCLUSIONS  97 

TKere  is  no  functional  activity  without  the  simultaneous 
consumption  of  the  nervous  material.  In  the  nerve, 
then,  irritability  can  be  measured  by  the  increased 
metabolism  which  occurs  on  stimulation.  If  the  irrita- 
bility is  high,  the  carbon  dioxide  increment  is  also  large, 
and  vice  versa.  The  response  to  stimulation  is  the  sign 
of  irritability.  We  measure  this  response  by  measuring 
the  simultaneous  output  of  carbon  dioxide,  which  must 
be  the  sign  of  that  metabolic  activity  in  virtue  of  which ^> 
the  function  ^performed. 

In  chapter  iv  we  demonstrated  further  the  importance 
of  the  metabolic  changes  for  the  functional  activities  of  ~ 
the  nerve,  and  we  hinted  that  the  real  mechanism  which 
makes  the  nerve  able  to  perform  its  function  must  be 
the  chemical  changes  which  go  on  in  the  resting  con- 
dition of  the  nerve,  and  which  must  determine  not  only 
the  degree  of  excitability  of  the  nerve — the  direction  of 
the  nerve  impulse — but  also  how  fast  this  transmission 
travels.  /We  found,  for  example,  that  the  part  of  the 
nerve  from  which  the  nerve  impulse  normally  conies 
always  produces  more  carbon  dioxide  than  the  part 
toward  which  it  is  going.  \  It  is  well  known  that  nerves 
are  more  excitable  in  the  parts  which  normally  originate 
the  impulse  and  that  the  excitability  decreases  down 
the  fiber.  Thus  there  is  a  parallelism  between  the 
degree  of  excitability  and  the  amount  of  carbon  dioxide 
produced  in  different  parts  of  the  same  fiber,  a  parallelism 
the  profound  importance  of  which  is  not  easily  over- 
looked. For  nerves  are  thus  shown  to  have  in  them  a 
metabolic  gradient.  They  are,  as  it  were,  polarized 
metabolically,  and  thus  we  have  for  the  first  time  the 
explanation  of  the  electrical  current  which  has  «been 


g8  A  CHEMICAL  SIGN  OF  LIFE 

found  to  run  in  nerves  between  the  two  cut  ends  of  the 
nerve,  up  or  down  it,  as  the  case  may  be.  Evidently 
electrical  current  is  generated  in  a  nerve  between  two 
parts  which  are  unequally  irritable,  or  unequally  under- 
going chemical  change.  The  part  of  the  nerve  which 
is  respiring  most  is  in  a  different  electrical  state  from  that 
which  is  respiring  less,  and  thus  we  see  the  very  clear 
and  definite  relationship  between  the  chemical  and  the 
electrical  changes  which  have  been  particularly  dwelt 
upon  by  Waller.  This  is  one  of  the  most  important  and 
fundamental  discoveries  which  we  have  noted,  for  it 
means  that  there  must  be  a  decrement  in  the  rate  of  the 
impulse  as  it  flows  down  the  fiber,  and  that  the  distance 
to  which  a  nerve  impulse  can  be  transmitted  is  not 
indefinite,  but  that  that  impulse  diminishes  as  it  pro- 
ceeds, and  will  ultimately  die  out.  In  the  medullated 
nerves,  to  be  sure,  this  decrement  is  not  large,  for  it  is 
very  necessary  that  it  should  be  as  small  as  possible  in 
the  more  highly  developed  nerves;  but  it  is  to  be  found 
everywhere.  And  in  simple  undifferentiated  proto- 
plasm of  plants  and  animals  it  is  easily  shown  to  exist. 
The  more  rapidly  nerves  respire  the  faster  do  they 
appear  to  carry  the  impulse;  irritability  and  the  rate 
of  production  of  carbon  dioxide  in  resting  nerves  thus 
appear  to  be  correlated.  The  more  respiration  the 
more  life!  If  we  abolish  respiration  temporarily,  or 
reduce  it,  we  find  that  irritability  has  been  reduced  in 
somewhat  the  same  proportion.  Anesthetized  nerves 
of  all  kinds  show  a  reduced  output  of  carbon  dioxide, 
and  they  recover  their  irritability  when  they  breathe 
again.  Anesthetics  do  not,  therefore,  affect  the  physical 
state  of  the  protoplasm  only,  as  they  have  been  supposed 


CONCLUSIONS  99 

to  do  by  many,  but  their  action  is  shown  by  the  change 
in  respiration  in  a  manner  more  perfect  than  in  any 
other  way  except  by  the  electrical  response.  Small 
amounts  of  anesthetics  at  first  increase  irritability;  and 
at  first  they  increase  the  rate  of  respiration  and  coinci- 
dently  they  increase  the  electrical  response.  Irritability, 
respiration,  and  electrical  response  parallel  each  other  so 
completely  that  they  are  evidently  different  aspects  of 
the  same  thing. 

In  chapter  v  what  we  had  established  as  being  true 
in  the  case  of  nerves  was  shown  to  be  true  in  the  case 
of  all  forms  of  living  matter.  Taking  the  least  promising 
kind  of  living  matter,  that  of  a  dry  seed,  we  demonstrated 
that  it,  too,  breathed  as  long  as  it  lived,  that  it 
produced  carbon  dioxide,  and  increased  its  output 
of  carbon  dioxide  when  it  was  mechanically  stimu- 
lated by  being  crushed.  Seeds,  too,  it  was  shown, 
could  be  anesthetized,  in  which  condition  they  give  off 
less  carbon  dioxide  and  no  longer  respond  by  an  outburst 
of  carbon  dioxide  when  injured.  Extending  our  observa- 
tions, we  found  that  all  kinds  of  plant  and  animal  tissues, 
without  any  exception,  respond  in  a  manner  similar  to 
that  of  the  nerve  fiber.  In  all  cases  stimulation  causes 
an  increase  in  carbon  dioxide.  We  could  never  find 
any  response  unaccompanied  by  an  outburst  in  car- 
bon dioxide.  Hence  the  best  way  to  discover  whether 
a  tissue  is  living  is  to  crush  it  and  see  whether  it  reacts 
to  the  injury  by  producing  more  carbon  dioxide.  It  is 
not  necessary  to  put  seeds  in  the  ground  to  determine 
whether  they  live;  by  crushing  some  of  them  we  may 
discover  whether  they  are  alive  or  not.  Thus  the 
chemical  test  of  life  in  the  tissues,  a  test  which  parallels 


ioo  A  CHEMICAL  SIGN  OF  LIFE 

at  every  point  Waller's  electrical  test,  is  shown  to  be 
whether  or  not  the  tissue  respiration  can  be  accelerated 
by  an  injury.  And  we  can  measure  this  with  our  new 
apparatus,  the  biometer,  which  thus  justifies  its  name, 
although  its  applicability  is  far  greater  than  merely 
testing  the  degree  of  vitality  of  a  tissue. 

We  have  now  to  compare  for  a  moment  this  criterion 
of  life — the  chemical — with  other  criteria  which  have 
been  proposed,  and  to  see  whether  it  lacks  anything  of  the 
precision  of  these  other  methods,  and  whether  life  can  be 
shown  to  exist  by  other  methods  where  we  cannot  prove 
its  existence  by  ours.  There  is  one  criterion  other  than 
the  obvious  one  of  growth  which  has  been  proposed  to 
determine  whether  a  seed  or  other  living  thing,  or  piece 
of  a  living  thing,  is  alive  or  not.  That  is  the  criterion 
suggested  by  Waller.  It  is  the  electrical  sign  of  life. 
Waller  discovered  a  very  remarkable  electrical  sign  of 
life,  which  may  be  described  as  follows :  Two  electrodes 
are  placed  on  opposite  sides  of  a  garden  pea  which  is 
living,  the  electrodes  being  connected  on  the  one  hand 
with  an  induction  coil  and  on  the  other  with  a  sensitive 
galvanometer.  A  single  induction  shock  is  then  sent 
through  the  pea.  If  the  pea  is  alive,  this  shock  is  fol- 
lowed by  a  remarkable  outburst  of  electromotive  force 
in  the  pea.  A  current  suddenly  blazes  out,  as  is  shown 
by  the  deflection  of  the  galvanometer.  It  is  as  if  the 
pea  jumped  when  stimulated.  This  current  sometimes 
travels  in  the  same  direction  as  the  induction  shock, 
and  sometimes  in  the  opposite  direction.  It  is  of  momen- 
tary duration.  Waller  calls  it  the  blaze  current.  As 
long  as  the  seed  lives,  you  get  it;  when  the  seed  dies,  you 
do  not  get  it.  The  dead  or  anesthetized  seed  does  not 


CONCLUSIONS  'ci 

blaze  up.  It  no  longer  reacts  to  a  stimulus.  Nearly  all 
kinds  of  tissues  show  these  blaze  currents  as  long  as 
they  are  alive,  and  the  outburst  is  not  only  a  sign  of  life, 
but  an  index  of  the  amount  of  life.  Life  and  electricity 
are  inextricably  bound  up  together.  In  the  sea  algae 
alone  Waller  failed  to  find  this  blaze  current,  but  he  does 
no,t  doubt  that  it  exists  there.  One  does  not  obtain 
it  for  the  reason,  probably,  that  the  salts  of  the  sea-water 
close  the  current  through  the  tissue  rather  than  through 
the  galvanometer.  Perhaps  a  low-resistance  galva- 
nometer would  detect  it  here  too.  This  sign  of  life  of 
Waller  is  in  many  ways  the  most  convenient  that  we 
.have,  if  only  we  have  the  apparatus  for  the  detection 
of  these  currents  ready  at  hand  and  set  up  for  use. 

But  up  to  this  point  we  were  still  in  the  dark  regarding 
the  cause  of  this  electrical  response.  We  could  not  know 
whether  it  was  due  to  a  physical  or  a  chemical  change 
in  the  tissues.  It  might  be  due  to  some  change  of 
permeability  of  the  tissues,  or  it  might  be  due  to  a  chem- 
ical change.  Waller  believed  it  to  be  caused  by  the 
latter,  and  his  conclusion  was  undoubtedly  correct. 

Waller  also  observed  that  following  this  electrical 
display  there  was  a  sudden  lowering  in  the  electrical 
resistance  of  the  pea  or  other  tissue.  This  might 
also  be  called  a  sign  of  life,  but  it  is  not  so  clear  and 
striking  as  the  blaze  current.  Evidently  this  is  by  no 
means  so  reliable  a  sign  of  life  as  the  other.  The  de- 
creased resistance  might  be  due  to  a  physical  change  of 
state  of  the  protoplasm  or  of  the  membranes,  so  that  the 
salt  solution  became  more  continuous;  or  it  might  be 
due  to  the  stimulation  increasing  in  some  way  the  ions  in 
the  protoplasm.  It  is  impossible  to  say  which. 


io  i  A  CBSMJCAL  SIGN  OF  LIFE 

The  chemical  sign  of  life  which  we  now  propose  for 
acceptance  is  in  many  ways  more  fundamental  than  the 
electrical.  It  is  probable,  as  Waller  suggested,  that 
the  chemical  changes  underlie  and  produce  the  electrical, 
and  they  produce  the  functional  changes,  such  as  the 
movements  which  follow  the  excitation.  In  the  chemical 
changes,  then,  we  seem  to  be  dealing  with  something 
more  fundamental  than  when  dealing  with  the  electrical, 
although,  if  we  admit  that  all  processes  of  oxidation  are 
in  reality  electrical,  this  distinction  cannot  be  sustained. 
Wherever  Waller  has  been  able  to  show  the  electrical 
sign  of  life,  we  can  show  the  chemical  sign,  and  we  can 
show  life  at  some  points  where  he  could  not,  as  in  the 
case  of  the  sea  algae.  These,  under  our  method,  respond 
in  the  same  manner  as  do  all  other  forms  of  living  matter. 
Moreover,  we  can  use  this  method  where  it  is  impos- 
fcible  to  use  the  electrical;  for  example,  in  very  minute 
forms  of  living  things,  like  eggs  of  small  size,  bacteria, 
or  infusoria.  Our  method  can  make  it  clear  that  they 
are  alive  and  breathing  and  responding  to  changes  in 
their  environment  like  every  other  living  thing.  It 
appears,  then,  that  this  sign  of  life  has  also  certain 
virtues  of  its  own,  although  it  is  not  so  striking  and 
elegant  as  the  method  of  Waller.  It  is  also  not  so  easy, 
perhaps,  for  the  ordinary  man  to  set  up  and  work 
this  apparatus  as  a  galvanometer.  But  what  it  lacks 
in  ease  it  makes  up  in  precision,  in  the  quantitative  nature 
of  its  results,  and,  above  all,  in  its  fundamental  char- 
acter. By  it  we  get  as  near  as  we  have  yet  got  to  life 
itself. 

In  still  another  way  the  results  which  are  recorded 
here  are  of  a  most  fundamental  character,  for  one  of  the 


CONCLUSIONS  103 

most  interesting  problems  of  general  physiology  has  been 
to  determine  what  is  the  nature  of  the  irritable  response 
which  living  matter  shows.  It  is  this,  the  problem  of 
problems,  which  we  wish  to  have  solved.  Is  that 
process  physical  or  chemical?  Is  it  simply  an  altera- 
tion of  permeability  of  membranes,  as  some  have 
supposed,  or  is  it  in  reality  in  the  nature  of  an  explosion  ? 
Is  the  living  thing  essentially  a  bag  of  jelly  with  a 
wonderful  membrane  about  it,  that  membrane  being  so 
wonderful  that  all  the  phenomena  of  life  are  to  be 
ascribed  to  its  changes  in  state?  For  this  is  the  view 
which  some  maintain.  They  lead  us  to  the  holy  of 
holies  of  cells  and  tell  us  to  behold  a  membrane!  Is  life 
nothing  more  than  a  membrane  ?  What  kind  of  a  subter- 
fuge is  this  which  we  encounter  ?  All  the  riddles  of  life 
are  but  the  peculiar  properties  of  a  membrane!  Upon 
this  membrane,  as  upon  a  magic  carpet  of  Arabia,  We 
are  invited  to  mount  and  travel  over  that  unexplored 
country  whose  mountain  peaks  shine  in  the  distance. 
Are  we,  then,  beings  of  but  two  dimensions,  nothing 
but  membranes,  of  which  the  magic  proportions  mock 
us  derisively,  since  we  can  never  hope  to  seize  that 
which  has  but  two  dimensions?  That  such  a  view 
resembles  the  membrane  it  has  conjured  up,  in  that  it  is 
surface  without  depth,  is  self-evident. 

In  no  such  simple  and  naive  a  manner  can  the  un- 
knowns in  the  equation  of  life  be  determined.  For  we 
have  found  that  everywhere,  paralleling  the  irritability 
changes  in  a  perfect  degree,  as  far  as  we  have  been  able 
to  determine,  go  the  chemical  changes.  Carbon  dioxide, 
that  very  simple  substance,  the  last  term  in  the  katabo- 
lism  of  living  matter,  rises  and  falls  with  irritability. 


104  A  CHEMICAL  SIGN  OF  LIFE 

Function  without  chemical  change  has  been  found  no- 
where. Respiration,  or  at  least  this  phase  of  respira- 
tion, and  irritability  are  in  some  way  bound  up  together, 
and  we  many  now  very  briefly  ask  ourselves  how  they 
may  be  related. 

The  connection  between  irritability  and  metabolism.— 
What,  then,  is  the  connection  between  the  irritable  and 
the  respiratory  process?  Could  we  answer  this  ques- 
tion we  should  have  solved  one  of  the  most  fundamental 
of  all  questions  of  science.  However,  we  do  not  hope 
to  be  able  to  answer  it  at  present.  But  let  us  at  least 
see  what  facts  we  can  discover.  The  first  of  these  facts 
which  strikes  us  is  that  living  matter,  even  when  it  is 
not  stimulated,  continues  to  give  off  carbon  dioxide 
and  to  respire.  What  is  the  significance  of  this  fact? 
Why  should  this  constant  consumption  of  material  go 
on  in  the  absence  of  outside  work  to  do  ?  The  main 
function  of  the  resting  metabolism  is  to  keep  the  tissue 
irritable.  As  long  as  a  tissue  remains  living  and  irritable 
we  find  it  to  be  the  seat  of  production  of  carbon  dioxide. 
To  be  sure,  it  continues  sometimes  to  give  off  carbon 
dioxide  after  death,  but  it  never  ceases  to  do  so  as  long 
as  it  is  alive.  After  death  the  rate,  with  possibly  a  tem- 
porary increase,  soon  diminishes.  For  nothing  is  more 
certain  than  that  living  matter  burns  up  faster  than 
the  same  matter  after  death.  Death  extinguishes  the 
torch  of  life,  although  it  may  continue  to  smoulder  for  a 
time  when  the  spirit  of  its  flame  is  gone. 

Does  not  this  fact  mean  that  life,  or  rather  the  living 
state,  is  a  dynamic  rather  than  a  static  phenomenon  ? 
We  might  conceive  the  living  matter  as  a  very  highly 
explosive  substance,  very  unstable  and  ready  to  go  to 


CONCLUSIONS  105 

pieces  under  a  very  slight  stimulus.  It  has  often  thus 
been  represented.  But  this,  after  all,  is  a  static  view. 
We  have  discovered  this  very  fundamental  fact,  that 
the  resting  metabolism  goes  faster  when  the  tissue  is 
more  abounding  in  life  and  is  more  irritable.  It  is  the 
burning  substance  which  is  irritable,  or,  perhaps,  the 
carbon  dioxide  thus  formed  conditions  in  some  way 
the  vital  or  irritable  reaction.  It  requires  the  expendi- 
ture of  energy  to  keep  living  matter  in  an  irritable  state. 
The  reaction  is  dynamic  and  not  static.  Living  matter 
has  been  conceived  by  many  physiologists  (of  whom  I 
may  mention  only  one  of  the  leading  exponents,  Verworn, 
for  the  various  modifications  of  this  view  of  individual 
authors  are  not  fundamental  modifications)  as  being 
composed  of  very  complex  unstable  molecules,  or  aggre- 
gates of  molecules  which  are  very  unstable.  These 
are  called  biogens.  Now  this  view  is  essentially  static. 
There  is  no  reason  why  a  biogen  should  not  be  isolated 
if  our  methods  were  but  fine  enough.  There  is  no  reason 
why  a  collection  of  biogens  should  not  exist  without  any 
metabolism;  why,  in  other  words,  suspended  anima- 
tion should  not  be  possible.  But  the  facts  which  we 
have  discovered  of  the  parallelism  of  the  production 
of  carbon  dioxide  and  irritability  lend  support,  it  would 
seem,  to  the  dynamic,  rather  than  to  the  static,  view. 

We  can  picture  the  process,  perhaps,  in  the  following 
crude  and,  of  course,  indefinite  manner:  The  life- 
process  may  be  considered  as  a  bicycle  in  motion.  The 
living  process  is  an  unstable  condition.  It  is  like  a 
chemical  system,  the  system  as  a  whole  having  a  certain 
stability,  but  being  at  the  same  time  the  seat  of  intense 
chemical  change.  It  is  in  an  unstable  equilibrium.  The 


io6  A  CHEMICAL  SIGN  OF  LIFE 

bicycle  running  rapidly  will  not  fall  down.  The  process 
of  standing  up  may  be  compared  to  the  vital  process 
in  the  resting  state.  The  degree  of  irritability  will  be 
measured  by  the  force  necessary  to  change  the  angle  it 
makes  with  the  ground.  Perhaps  the  amount  of  life  may 
be  compared  to  the  angle  the  bicycle  makes  with  the 
ground.  The  metabolic  activity  is  the  force  which 
moves  the  wheel.  The  locomotion  of  the  wheel  is  the 
functional  activity.  There  is,  however,  this  difference— 
the  faster  the  bicycle  moves  the  more  stable  it  is,  whereas 
the  faster  the  respiration  drives  the  less  stable  is  the 
irritability  of  the  tissues.  Our  simile  breaks  down  here. 
And  indeed  it  is  but  a  poor  picture,  of  not  much  value. 
But  whatever  view  we  may  take  of  the  matter,  we  may 
at  least  be  sure  of  this  much :  that  chemical  change  is  in- 
volved in  irritability.  The  transmission  of  a  nerve 
impulse  involves  material  decomposition  in  the  fiber. 
The  impulse  may  be  nothing  else  than  the  increased 
metabolism  itself.  The  nerve  impulse  is  a  very  real 
thing,  and  it  has  a  material  basis  which  we  may  hope  to 
discover.  So  far  we  have  found  two  facts  about-  it: 
first,  it  liberates  carbon  dioxide  as  it  passes  over  the 
fiber;  and,  second,  it  depends  on  the  nerve  fiber  having 
been  previously  oxidized  or  exposed  to  oxygen.  Evi- 
dently combustion  is  involved  in  the  process  somewhere, 
but  it  appears  at  present  more  probable  that  it  is  involved 
in  the  creation  of  the  irritable  substance  rather  than  in 
the  very  act  of  excitation  itself.  In  other  words,  the 
oxidation  is  part  of  the  process  of  repair  or  the  recovery 
of  the  tissue — the  process  by  which  the  state  of  irrita- 
bility is  maintained — and  not  the  process  of  transmission 
of  the  impulse  itself. 


CONCLUSIONS  107 

^Concerning  the  nature  of  the  material  basis  of  the 
nerve  impulse  we  can  only  say  that  it  appears  to  involve 
that  part  of  the  chemical  transformations  in  protoplasm 
which  result  in  the  production  of  carbon  dioxide. 
Farther  than  this  we  cannot  go  at  present.  But  it  is 
certain  that  it  has  a  chemical  basis.  Whether  it  has  also 
a  physical  basis,  such  as  a  change  in  state  of  the  colloidal 
substratum  of  the  nerve,  or  not,  we  cannot  yet  say. 
Who  shall  write  the  chemical  reaction  of  the  future, 
embracing,  not  only  the  energy  exchange,  but  the  change 
in  psychism  as  well  ? 

Finally,  we  come  to  the  quantity  of  life,  the  point 
from  which  we  started.  The  measure  of  this  is  the 
amount  of  respiration,  or  the  amount  of  electrical 
response  shown  on  stimulation.  The  question  of  how 
much  we  are  alive  must  be  answered  by  the  determina- 
tion of  the  extent  to  which  we  are  undergoing  energy 
transformation.  Death  and  peace,  life  and  struggle — 
these  are  the  pairs  which  go  together.  The  most  perfect 
young  life  is  that  which  shows  the  highest  metabolic 
rate.  We  have  shown  the  general  correlation  between 
the  carbon  dioxide  production  and  the  nerve  impulse 
in  its  speed  of  propagation  and  ease  of  origin.  There 
must,  then,  be  a  close  correspondence  between  the  habit 
of  the  organism  and  the  general  metabolic  rate.  The 
simile  of  the  torch  is  obvious.  The  faster  it  burns  the 
more  light  and  life  it  has.  The  most  vigorous  life  is 
that  with  the  keenest  chemical  change.  And  this  is  also, 
as  has  been  shown  in  another  volume  in  this  series,  the 
criterion  of  youth.  The  most  successful  life  is  that  in 
which  the  nervous  system  remains  active,  youthful,  and 
alive  for  the  greatest  number  of  years.  It  is  the  youth- 


io8  A  CHEMICAL  SIGN  OF  LIFE 

ful,  keenly  metabolic  nervous  system  which  is  most 
responsive  to  its  environment. 

Whatever  may  be  the  nature  of  that  activity  going  on 
in  our  minds,  we  have  at  least  discovered  something 
about  its  simplest  chemical  accompaniment.  Perhaps 
the  nerve  impulse  is  something  in  the  nature  of  a  prop- 
agated explosive  wave  in  a  continuous  substance. 
Whether  that  wave  is  in  the  nature  of  a  hydrolysis  or  an 
oxidation  we  cannot  say,  but  at  any  rate  it  results  in  the 
liberation,  in  some  manner,  of  carbon  dioxide.  This 
substance  tells  us  whether  the  nerve  impulse  has  passed 
this  way  or  not.  The  change  which  liberates  it  may  be 
the  impulse  itself.  Three  kinds  of  changes  occur,  then, 
in  our  brains  when  the  nerve  impulses  are  passing — an 
electrical  change,  a  chemical  change,  and  a  psychical 
change.  Which  is  the  fundamental  change? 


APPENDIX 
THE  BIOMETER:   HOW  TO  USE  IT 

The  study  of  carbon  dioxide  has  been  so  connected 
with  various  forms  of  human  activity  that  in  spite  of 
natural  difficulties  methods  for  its  accurate  quantitative 
determination  have  been  highly  developed.  Never- 
theless, none  of  the  various  methods  of  analysis  can  be 
used  for  very  minute  quantities  of  carbon  dioxide.  The 
greatest  difficulty  in  using  any  micro-gas  analysis  is  in 
securing  freedom  from  the  external  variations  of  tempera- 
ture and  pressure.  Particularly  is  this  so  in  the  case  of 
carbon  dioxide,  for  we  need  to  consider,  not  only  the 
effect  of  temperature  and  pressure  variation,  but  also 
how  to  free  the  apparatus  from  atmospheric  carbon 
dioxide.  After  we  discovered  a  new  method  which 
detected  exceedingly  minute  quantities  of  this  gas  we 
found  that  the  ordinary  method  of  freeing  air  or  any 
other  gases  from  carbon  dioxide  was  not  sufficiently 
accurate,  although  it  should  be  admitted  that  our 
experience  in  washing  gases  was  not  very  extensive. 
The  biometer  is  constructed  with  a  view  to  meeting 
these  difficulties  and  has  shown  itself  to  be  remarkably 
convenient  for  many  biological  as  well  as  chemical 
investigations. 

Uses  of  biometer. — The  biometer  can  detect  carbon 
dioxide  in  as  small  quantities  as  one  ten-millionth  of  a 
gram.  This  is  the  amount  contained  in  one-sixth  of  a 
cubic  centimeter  of  the  purest  air,  in  which  we  assume 

109 


no  A  CHEMICAL  SIGN  OF  LIFE 

3.0  parts  of  carbon  dioxide  present  in  10,000.  The 
delicacy  of  the  apparatus  can  be  illustrated  by  the  kind 
of  experiments  we  can  use  it  for,  e.g. : 

1 .  The  different  rates  at  which  carbon  dioxide  is  pro- 
duced by  a  single  fertilized  and  a  single  unfertilized  egg 
of  a  fish  (Fundulus  hectroclitus)  can  be  distinguished. 

2.  The  unequal  rate  of  metabolism  of  two  different 
species  of  the  little  banana  flies  can  be  detected  within 
ten  minutes  by  using  a  single  insect  in  each  chamber. 

3.  The  vitality  of  a  single  kernel  of  wheat  can  be 
detected  in  ten  minutes. 

4.  The  daily  variation  of  respiratory  activity  of  a 
single  isopod  has  been  determined. 

5.  The  carbon  dioxide  production  of  the  different 
parts  of  a  small  nerve  fiber  can  be  measured,  and  the 
unequal  rates  of  different  segments  of  the  nerve  detected. 

6.  The  effect  on  the  metabolic  rate  of  the  muscular 
contractions  of  very  small  animals,  like  a  worm  or  insect, 
or  the  effect  of  light  on  small  pieces  of  a  leaf,  can  be 
demonstrated  in  the  class  in  a  few  minutes. 

7.  By  the  use  of  proper  reagents  small  quantities  of 
many  other  gases  can  be  measured. 

Principle  of  the  method. — The  principle  of  the  method 
was  first  devised  in  conjunction  with  Dr.  H.  N.  McCoy, 
and,  with  some  modifications,  the  biometer  is  constructed 
so  as  to  conform  thereto.  The  principles  involved  are  as 
follows : 

1.  Exceedingly  minute  quantities  of  carbon  dioxide 
can  be  precipitated  as  barium  carbonate  on  the  surface 
of  a  small  drop  of  barium  hydroxide  solution. 

2.  When  the  drop  of  barium  hydroxide  is  exposed  to 
any  sample  of  a  gas  free  from  carbon  dioxide  it  remains 


THE  BIOMETER:    HOW  TO  USE  IT  in 

clear,  but  when  more  than  a  definite  amount  of  carbon 
dioxide  is  introduced,  a  precipitate  of  carbonate  appears, 
which  is  detectible  by  means  of  a  lens. 

3.  By  the  use  of  accurately  known  quantities  of 
carbon  dioxide  of  exceedingly  high  dilution  it  was  found 
that  the  minimum  amount  of  carbon  dioxide  which  gives 
a  precipitate  is  i.oXio"7  g. 

4.  By  determining,  therefore,  the  minimum  volume 
of  any  given  sample  of  the  gas  necessary  to  give  the 
first   visible    formation   of  the  precipitate  its  carbon 
dioxide  content  can  be  estimated  accurately,  since  this 
volume  must  contain  just  the  known  detectible  amount 
of  the  gas,  which  we  found  to  be  i.oXio"7  g. 

5.  By  having  two  chambers  side  by  side  the  different 
rates  of  metabolism  from  two  different  tissues  can  be 
estimated  by  the  different  speeds  of  formation  of  the 
precipitate  and  extent  of  the  precipitate. 

Description  of  the  apparatus. — The  biometer  shown 
in  Figs,  i  and  3  is  made  of  glass.  It  consists  of  two 
respiratory  chambers  connected  by  a  three-way  stop- 
cock £,  the  other  arm  of  which  is  connected  to  one  arm 
of  another  three-way  stopcock  K.  (As  is  shown  in 
Fig.  i,  for  an  ordinary  experiment  we  can  connect  it 
directly  to  the  nitrometer.)  Each  of  the  other  two 
arms  of  stopcock  K  is  connected  to  a  nitrometer,  W  or 
X,  which  is  used  for  removing  the  final  traces  of  carbon 
dioxide  from  the  gas  with  which  the  chambers  are  to 
be  filled.  The  nitrometer  on  the  right  is  connected  to  a 
carboy  F  (see  Fig.  5,  apparatus  III),  filled  with  air  free 
from  carbon  dioxide ;  and  the  other,  on  the  left,  to  a  simi- 
lar carboy  as  a  reservoir  for  any  other  gases  that  may  be 
used  as  a  special  medium  for  different  experiments,  such 


H2  A  CHEMICAL  SIGN  OF  LIFE 


FIG.  i.— The  Biometer.     One- fourth  actual  size 


THE  BIOMETER:    HOW  TO  USE  IT  113 

as'  oxygen-free  air,  hydrogen,  or  volatile  anesthetics,  and 
which  are  exposed  to  an  alkaline  solution  in  order  that 
every  trace  of  carbon  dioxide  can  be  removed  from  the 
gas  in  question.  As  stated  before,  for  ordinary  metabo- 
lism experiments  we  may  use  but  one  nitrometer  for 
ordinary  air  only,  as  shown  in  the  photograph  (Fig.  i). 
Chamber  A  is  drawn  to  a  capillary  stopcock  C;  chamber 


FIG.  3. — Biometer.  One-third  actual  size.  The  shaded  portions 
of  the  apparatus  indicate  the  rubber  connection,  which  is  first  coated  by 
shellac  and  then  sealed  with  a  special  sealing  wax.  Some  parts  are 
also  sealed  with  mercury. 

B  is  drawn  to  a  similar  capillary  stopcock  C',  one  arm 
of  which  is  connected  to  another  three-way  stopcock  G, 
one  arm  of  which  is  connected  to  a  mercury  burette  T, 
which  is  used  for  adjusting  the  pressure  in  the  apparatus. 
(The  slightly  different  structures  should  be  noted  here 
in  Figs,  i  and  3,  the  latter  having  no  capillary  stopcock 
C,  but  being  directly  connected  with  the  three-way 
stopcock  G.  As  the  latter  apparatus  requires  consider- 
able experience  in  order  to  make  it  perfectly  air-tight, 
the  former  type  only,  as  shown  in  the  photograph,  is 


H4  A  CHEMICAL  SIGN  OF  LIFE 

furnished  by  Eimer  &  Amend  when  the  biometer  is 
ordered.)  Each  of  the  chambers  has  a  capacity  of  20 
to  25  c.c.  and  is  provided  with  the  platinum  electrodes 
n  and  m  for  stimulation  purposes,  and  also  with  a 
glass  stopper  S  or  R,  which  can  be  sealed  with  mercury. 
The  air  pump  is  connected  through  /  and  the  barium 
hydroxide  solution  is  introduced  through  V  to  d  and  /, 
where  the  drops  are  to  be  formed. 

How  to  set  up  the  biometer. — In  order  to  get  up  this 
apparatus,  the  following  materials  will  be  necessary: 
one  biometer  proper;  two  ordinary  three-way  stopcocks; 
one  nitrometer;  one  ordinary  glass  stopcock;  one 
water  pump;  one  mercury  burette,  made  of  four  or  five 
inches  of  any  broken  burette;  one  bottle  with  a  side 
neck  at  the  bottom  of  about  300  c.c.  capacity  (aspirator 
bottles);  two  large  carboys;  one  capillary  "["-tube  to  be 
bent  to  fit  the  biometer  proper  at  Q  and  F;  three 
pinchcocks;  one  empty  acid  bottle  for  a  half -saturated 
solution  of  barium  hydroxide;  two  CaCL  tubes  or  wash- 
bottles  to  protect  barium  hydroxide,  and  one  carboy; 
one  lo-pound  can  of  Greenbank  alkali;  500  g.  of  C.P. 
barium  hydroxide;  one  yard  of  thick- walled  pressure 
tubing;  one  yard  of  good  antimony  tubing  to  fit  the 
ordinary  glass  tubing;  a  little  sealing  wax;  200  c.c.  of 
redistilled  mercury;  a  few  yards  of  glass  tubing  of  or- 
dinary sizes. 

Excepting  for  the  biometer  proper,  most  of  the 
materials  mentioned  above  can  be  found  in  an  ordinary 
laboratory  or  can  be  substituted  with  homemade  appa- 
ratus without  losing  the  accuracy  of  the  method. 

With  these  materials  on  hand,  the  apparatus  can  be 
set  up  without  any  difficulty  if  one  follows  the  figure  very 


THE  BIOMETER:    ROW  TO  USE  IT  115 

cldsely.  The  best  way  to  set  it  up  is  to  mount  it  per- 
manently, instead  of  clamping  it  with  several  iron 
clamps  and  stands.  The  apparatus  set  up  as  in  Fig.  i 
in  the  lead  frame  or  in  the  wooden  frame  not  only  looks 
better,  but  is  subject  to  less  damage. 

How  to  clean  the  apparatus. — The  apparatus  is  con- 
structed and  is  mounted  in  such  a  way  that  washing  and 
cleaning  can  be  done  after  each  experiment  without 
taking  it  apart.  Although  the  procedure  of  washing 
is  exceedingly  simple,  it  is  better  for  the  beginner  to 
follow  exactly  the  directions  given  below,  for  there  are 
many  stopcocks  which  have  to  be  turned  in  a  particular 
direction  in  order  to  avoid  unnecessai^accidents  which 
sometimes  necessitate  the  expenditure  of  considerable 
time  in  bringing  the  apparatus  back  into  a  working  con- 
dition. An  example  will  illustrate  this:  If  one  forgets 
to  turn  the  stopcock  L  during  the  washing,  and  the 
space  between  L  and  K  becomes  wet,  it  will  require 
about  five  or  six  hours  to  wash  the  space  and  clean  and 
dry  it.  The  old  saying  that  an  ounce  of  prevention  is 
worth  a  pound  of  cure  should  be  borne  in  mind  here. 

Turn  the  water  pump  on.  Now  turn  stopcock  E 
1 80°  to  the  right,  so  that  the  barium  hydroxide  solution 
is  entirely  out  of  connection  with  the  other  two  arms  of 
the  stopcock  E.  Remove  mercury  from  stoppers  S  and 
R  with  a  pipette,  and  then  remove  the  stoppers  S  and  R, 
and  tissues  if  there  are  any.  Turn  stopcock  L  in  such  a 
way  that  the  bore  inside  will  look  like  this  _L,  thus 
severing  the  connection  between  the  vertical  arm  of  L 
and  the  horizontal  arms.  Turn  on  stopcocks  /  and 
Q  and  F  in  order.  Turn  stopcock  G  90°  to  the  left. 
Withdraw  the  mercury  from  chamber  A  by  opening 


n6  A  CHEMICAL  SIGN  OF  LIFE 

stopcock  C  and  from  B  by  opening  the  corresponding 
stopcock.  Fill  both  chambers  A  and  B  with  water 
acidulated  with  nitric  acid  (not  more  than  i  per  cent), 
having  both  stopcocks  open,  then  with  distilled  water  five 
times,  then  once  with  alcohol,  and  once  with  alcohol-ether. 
Two  funnels  placed  under  these  two  chambers  (see  Fig.  i) 
are  connected  with  a  sink,  to  form  an  outlet  for  this 
waste  water.  The  alcohol  and  alcohol-ether  drained  out 
of  these  chambers  should  be  saved  for  re-use.  Replace 
the  stoppers  S  and  R.  Let  the  machine  remain  un- 
touched for  drying  while  the  suction  pump  is  going  and 
while  the  tissue  for  an  experiment  is  being  prepared. 
Five  or  ten  minutes  will  be  sufficient  for  complete  drying 
if  the  water  pump  is  in  good  order  and  the  alcohol  used 
has  not  contained  too  much  water. 

It  is  very  important  that  we  should  leave  the  appa- 
ratus in  this  condition  until  we  are  ready  for  an  experi- 
ment, and  that  no  stopcock  should  be  touched,  for  this 
is  the  only  condition  under  which  all  parts  of  the  appara- 
tus will  dry. 

How  to  obtain  air  free  from  carbon  dioxide. — It  is  very 
difficult  to  make  air  completely  free  from  carbon  dioxide 
by  the  ordinary  method,  i.e.,  by  merely  passing  it  through 
several  alkaline  bottles  or  alkaline  towers.  A  simpler 
and  surer  method  is  shown  together  with  apparatus  III  in 
Fig.  5  (p.  131).  It  is  prepared  by  shaking  air  with  a  20  per 
cent  solution  of  sodium  hydroxide  in  a  tightly  stoppered 
carboy  F,  supplied  with  suitable  tubes,  one  of  which  is 
led  to  another  carboy  E,  which  is  filled  with  about  10 
to  15  per  cent  alkali  solution.  When  the  air  in  carboy 
F  is  to  be  used,  it  is  driven  into  the  nitrometer  C  (appa- 
ratus III)  or  W  (in  case  of  the  biometer),  which  is  filled 


THE  BIOMETER:    HOW  TO  USE  IT  117 

with  a  less  concentrated  alkaline  solution  (a  weaker 
solution  is  necessary  so  that  the  chamber  may  be  filled 
with  air  which  is  not  too  dry).  Driving  the  air  into 
the  nitrometer  is  accomplished  either  by  increasing  the 
pressure  in  carboy  F,  by  introducing  more  alkali  from  the 
carboy  above  E,  or  by  introducing  more  alkali  through 
funnel  instead  of  from  another  carboy.  After  each 
evacuation  of  the  apparatus  by  a  strong  suction  this 
air  free  from  carbon  dioxide  is  introduced  from  the 
nitrometer  C  or  W  into  the  chambers  through  the  stop- 
cock I.  For  ordinary  experiments  one  can  keep  the 
pressure  in  the  carboy  F  high  enough  so  that  air  free 
from  carbon  dioxide  can  be  driven  into  the  nitrometer 
simply  by  opening  stopcock  9  after  each  evacuation. 

How  to  test  purity  of  air. — In  order  to  test  whether 
or  not  the  air  in  carboy  F  is  free  from  carbon  dioxide 
the  following  experiment  is  necessary.  It  may  be 
stated  that  in  all  ordinary  experiments  we  use  exactly 
the  same  manipulation  as  the  one  now  to  be  described. 
When  the  apparatus  is  perfectly  dry,  the  pump  being 
at  work,  open  stopcock  (or  pinchcock)  9  so  that  the 
nitrometer  is  filled  with  the  air  freed  of  carbon  dioxide 
from  carboy  F.  (If  no  bubbles  come  out  by  opening  this 
pinchcock  it  means  that  there  is  not  enough  pressure  in 
the  carboy.  In  that  case  open  pinchcock  8  to  let  more 
alkali  siphon  down  from  the  carboy  above  E,  until 
about  200  to  300  c.c.  of  air  can  be  obtained  by  opening 
pinchcock  9.) 

Shut  stopcocks  C  and  C  and  /.  If  the  pressure 
pump  is  strong  enough  and  all  the  joints  are  tight, 
chambers  A  and  B  should  be  under  a  strong  negative 
pressure  by  now,  so  that  when  you  open  stopcock  C 


n8  A  CHEMICAL  SIGN  OF  LIFE 

the  mercury  in  the  little  vessel  can  be  sucked  up  to  the 
mark,  thus  making  the  remaining  volume  of  the  left 
chamber  exactly  15  c.c.1  With  a  pipette  fill  the  mercury 
burette  T  with  mercury  to  the  mark,  open  stopcock  G 
90°  to  the  right,  open  stopcock  C'  very  gently  till 
mercury  falls  to  the  second  mark  in  burette  T,  which  is 
so  marked  that  by  introducing  this  amount  of  mercury 
the  remaining  volume  in  this  chamber  B  is  now  15  c.c. 
after  the  barium  hydroxide  is  introduced  to  the  top  of 
the  barium  hydroxide  tube  in  left  chamber  A .  (There- 
fore, by  introducing  mercury  to  this  mark,  chamber 
B  has  a  capacity  less  than  15  c.c.,  but  by  introducing 
barium  hydroxide  in  the  left  chamber  A ,  some  of  the 
mercury  will  be  pushed  back,  so  that  the  capacity  of  the 
right  chamber  B  is  now  finally  exactly  15  c.c.)  Now 
shut  stopcock  C'  (very  important).  With  a  pipette  add 
mercury  to  the  mercury  burette  till  the  level  of  mercury 
in  it  becomes  a  little  lower  than  that  of  the  mercury  in 
chamber  B.  Now  pull  out  the  core  of  stopcock  /,  so  as 

1  The  exact  volume  of  each  chamber  should  be  calibrated  once  for 
all.  If  this  is  done,  one  can  always  work  with  a  constant  volume  in 
both  chambers,  so  that  when  a  known  amount  in  cubic  centimeters  of 
mercury  is  introduced  so  as  to  bring  it  up  to  the  marks  in  the  chambers 
the  remaining  volume  will  always  be  the  same.  The  advantage  in 
having  both  chambers  equal  in  capacity  is  obvious.  One  of  our  appa- 
ratuses has  a  capacity  of  21 . 5  c.c.  in  chamber  A  and  22  c.c.  in  chamber  B. 
We  therefore  introduced  6.5  c.c.  into  A  and  marked  the  level  of  the 
mercury,  and  7  c.c.  into  B  and  did  likewise.  Thus  when  mercury  is 
introduced  up  to  the  marks,  both  chambers  have  the  same  remaining 
volume,  namely,  exactly  15  c.c.  A  little  error  in  calibrating  the  chamber 
A  is  not  very  serious,  as  this  chamber  is  used  for  the  analytic  purpose 
only,  while  the  biometer  is  to  be  used  as  a  quantitative  apparatus;  but 
the  chamber  B  must  be  calibrated  with  extreme  care,  and  each  intro- 
duction of  the  known  amount  of  the  mercury  must  be  done  accurately. 
This  can  be  accomplished  by  means  of  the  mercury  burette  T,  which  is 
well  calibrated  and  can  measure  off  any  known  amount  of  mercury  with 
a  high  degree  of  accuracy. 


THE  BIOMETER:    HOW  TO  USE  IT  119 

t6  admit  air  into  the  apparatus,  and  shut  it.  (Remove 
stoppers  5  and  R;  introduce  a  tissue  to  the  right  cham- 
ber B  if  performing  an  actual  experiment,  and  replace 
the  stoppers.)  Seal  the  stoppers  5  and  R  with  mercury. 
Turn  stopcock  L  180°  to  the  right,  so  that  three  arms  are 
now  in  communication.  Shut  stopcock  /  and  open 
/  very  carefully  and  shut  /.  (It  should  never  be 
opened  unless  the  nitrometer  contains  more  than 
40  c.c.  of  air  and  stopcock  J  is  shut.)  Open  /  and 
shut  /;  open  /  and  shut  /.  In  this  way  we  evacuate 
the  chambers  by  opening  /,  and  fill  them  up  with 
pure  air  by  opening  /.  This  process  of  washing  the 
apparatus  with  air  freed  from  carbon  dioxide  is  repeated 
at  least  five  times.  At  the  end  of  the  last  washing, 
having  stopcock  /  shut  and  /  opened,  shut  stopcocks  Q 
and  F.  Without  touching  stopcock  /  open  stopcock  / 
and  raise  the  safety  bottle  D,  so  that  the  pressure  inside  of 
the  apparatus  is  now  equal  to  that  of  the  atmosphere,  and 
then  shut  7.  -Open  stopcock  C;  the  mercury  in  the 
burette  T  should  not  move  if  the  previous  pressure 
adjustment  with  the  safety  bottle  D  and  nitrometer  is 
properly  done.  Shut  the  stopcock  /  so  as  to  cut  off 
suction;  turn  stopcock  E  to  right  90°,  so  that  the  space 
between  /  and  E  will  be  filled  with  barium  hydroxide; 
turn  it  90°  more  to  the  right,  so  as  to  fill  all  the  capillary 
T-tube  below  Q  and  F  with  the  clear  solution  of  barium 
hydroxide.  Open  stopcock  Q  very  gently  until  a  hemi- 
spherical drop  of  half-saturated  barium  hydroxide  is 
formed  at  d.  Then  shut  Q  and  make  a  similar  drop  at/ 
in  the  other  chamber.  Turn  stopcock  L  45°,  so  that  the 
connection  between  the  two  chambers  is  now  severed. 
Shut  stopcock  Cf .  If  the  air  is  completely  free  from  a 


120  A  CHEMICAL  SIGN  OF  LIFE 

minute  trace  of  carbon  dioxide,  the  drop  of  barium 
hydroxide  should  be  clear,  not  only  at  the  time  of 
introduction  of  the  drop  at  the  beginning,  but  also  after 
standing  for  several  hours,  having  not  a  single  granule 
of  the  precipitate  visible  with  a  lens. 

Since  the  main  point  of  accuracy  in  our  apparatus 
depends  on  having  the  air  free  from  carbon  dioxide — 
indeed,  it  is  the  most  difficult  part  of  the  manipulation 
of  the  biometer  to  have  good  air — particular  care  must 
be  taken  to  have  every  point  of  junction  perfectly  air- 
tight. The  points  most  susceptible  to  leaking  will  be  the 
stopcocks  and  the  mouth  of  the  carboy  where  the  air  is 
preserved.  A  strong  suction  is  essential  for  a  complete 
washing  of  the  apparatus  with  the  air  free-  of  carbon 
dioxide. 

Methods  for  the  qualitative  detection  of  carbon  dioxide 
production  in  the  tissue. — After  the  apparatus  is  cleaned 
and  dried  and  the  air  is  ascertained  to  be  pure  for  use, 
a  prepared  tissue  is  placed  on  a  cover-slide  or  the  glass 
plate  shown  in  Fig.  2  (p.  38)  and  introduced  into  the 
chamber  B,  no  tissue  being  put  in  the  left  chamber  A. 
The  detailed  method  is  exactly  the  same  as  the  one 
just  described.  After  both  chambers  are  closed  with  the 
stoppers  S  and  R  and  sealed  with  mercury,  the  apparatus 
is  washed  with  the  air  free  of  carbon  dioxide  in  the  usual 
manner.  Barium  hydroxide  solutions  are  introduced 
into  d  and  /,  forming  hemispherical  drops,  and  the  con- 
nection between  the  two  chambers  is  severed  by  turning 
the  stopcock  between  them  (L);  then  watch  the  drop 
with  a  lens.  If  the  air  is  free  from  carbon  dioxide,  the 
drop  in  the  left  chamber  ought  to  be  perfectly  clear, 
while  the  drop  in  the  right  chamber,  if  the  tissue  gives 


THE  BIOMETER:    HOW  TO  USE  IT  121 

off  carbon  dioxide,  will  not  be  coated  with  the  precipitate, 
but  will  have  on  its  surface  some  crystals  of  barium  car- 
bonate, which  becomes  more  heavily  precipitated  as  the 
respiration  goes  on.  By  repeating  the  same  experi- 
ments after  interchanging  the  chambers,  using  the  left 
for  the  tissue  and  the  right  for  a  blank,  itjvill  be  possible 
to  eliminate  any  possible  error  which  might  come  from 
some  technical  fallacy  characteristic  of  one  particular 
chamber.  For  a  casual  observer  the  initial  granule 
will  not  be  distinct  from  a  granular  spot  on  the  glass. 
The  granules,  however,  will  soon  increase  over  the  surface 
of  the  drop  and  will  gradually  collect  downward  at  the 
edge  of  junction  of  the  drop  with  the  glass  tubing. 
The  thick  band  of  white  precipitate  around  the  bottom  of 
the  drop  will  gradually  extend  toward  the  top  of  the 
drop,  so  that  after  the  band  reaches  more  than  half  of  the 
hemispherical  drop  of  barium  hydroxide  one  can  see 
with  the  naked  eye,  not  only  from  the  side,  but  also 
from  above,  the  whole  drop,  now  resembling  a  contract- 
ing iris.  When  the  very  top  of  the  drop  is  filled  with  the 
precipitate,  the  whole  drop  of  barium  hydroxide  will 
look  very  opaque,  covered  with  a  thin  layer  of  the  car- 
bonate. If  one  take  a  small  piece  of  sciatic  nerve  of  a 
frog,  say  about  20  mg.,  he  can  see  these  different  stages 
of  precipitation  very  distinctly,  but  when  the  amount 
of  tissue  taken  is  very  large  it  is  very  difficult  to  observe 
these  phenomena  on  account  of  the  too  rapid  formation 
of  the  precipitate  all  over  the  surface  of  the  drop.  It 
is  therefore  best  to  take  a  very  small  piece  of  the  nerve 
for  the  purpose  of  following  these  different  stages  of  the 
precipitation  of  carbon  dioxide  as  carbonate,  for  the 
practice  of  distinguishing  these  different  stages  is  very 


122  A  CHEMICAL  SIGN  OF  LIFE 

useful  for  a  quick  comparative  estimate  of  the  different 
rates  of  carbon  dioxide  production  from  two  different 
samples  of  tissue. 

Methods  for  a  quick  comparative  estimate  of  carbon 
dioxide  production  from  two  different  samples  of  tissue. — 
By  repeating  quantitative  experiments  it  was  found  that 
the  speed  with  which  the  first  precipitate  appears,  the 
sizes  of  the  precipitates,  and  the  shapes  of  the  aggrega- 
tion of  the  deposits  at  different  stages  represent  different 
quantities  of  carbon  dioxide,  if  compared  simultaneously 
under  the  same  conditions.  Thus,  with  this  remarkably 
simple  means  we  can  determine  quickly  the  comparative 
output  of  carbon  dioxide  from  two  different  tissues  at 
the  same  time.  The  method  of  procedure  is  best  illus- 
trated by  the  following  example: 

Two  pieces  of  the  sciatic  nerve  are  isolated  from 
the  same  frog  and  weighed  into  approximately  the  same 
mass.  One  piece  is  laid  on  one  glass  plate  and  the  other 
on  the  other  plate  in  such  a  way  that  one  part  of  the  nerve 
lies  across  the  electrodes  of  the  glass  plates  as  shown  in 
Fig.  2  (p.  38).  In  this  way,  when  the  plates  are  hung  on 
the  electrodes  n  and  m  either  nerve  desired  can  be  stimu- 
lated with  the  induction  current.  These  plates  are  now 
hung  on  the  electrodes  in  each  chamber,  and  the  usual 
procedure  is  followed  for  eliminating  carbon  dioxide  from 
the  apparatus.  After  the  connection  between  the  two 
chambers  is  closed  by  means  of  stopcock  L,  having  the 
drops  of  barium  hydroxide  in  each  chamber  as  usual, 
the  nerve  in  chamber  A  is  stimulated  by  the  current. 
Then  if  one  watches  over  the  surface  of  the  drops  care- 
fully from  the  start,  the  deposit  of  carbonate  will  be 
seen  to  appear  first  on  the  drop  in  chamber  A,  in  which 


THE  BIOMETER:    HOW  TO  USE  IT 


123 


the' stimulated  nerve  is  placed.  Later  the  total  amount 
of  the  precipitate  grows  much  larger  on  the  drop  in  this 
chamber.  This  clearly  shows  that  the  chamber  in  which 
the  larger  amount  of  the  precipitate  is  found  must  have 
the  higher  concentration  of  carbon  dioxide.  Since  we 
had  exactly  the  same  kind  of  air  at  the  beginning,  the 
conclusion  is  that  the  nerve  when  stimulated  must  give 
off  more  carbon  dioxide  than  the  resting  one.  This 
conclusion  can  easily  be  confirmed  by  exchanging  the 
nerve  in  the  chambers  as  usual. 

The  following  figures  will  illustrate  the  different  stages 
of  the  granulation  of  barium  carbonate  and  will  show 


i  23  4  5  50  o 

FIG.  4. — Different  stages  of  the  granulation  of  barium  carbonate  on 
the  surface  of  the  hemispherical  drop  of  barium  hydroxide;  $a  and  6a 
show  the  top  views  of  the  drops  at  the  time  when  "iris  effect"  is  pro- 
duced. 

definitely  how  easy  it  is  to  compare  the  amount  of  carbon 
dioxide  production  at  several  points.  And  such  com- 
parison can  be  confirmed  more  exactly  by  the  quantita- 
tive determination ;  the  details  of  the  method  are  given  in 
the  next  paragraph. 

The  method  for  quantitative  measurement  of  carbon 
dioxide. — While  the  apparatus  is  drying,  prepare  the 
tissue  and  weigh  it.  If  everything  is  perfectly  dry,  fill 
both  chambers  with  mercury  up  to  the  marks,  as  directed 
on  p.  118.  Remove  the  stopper  from  the  right  chamber 
only,  which  is  to  be  used  as  a  respiratory  chamber, 
and  the  tissue  is  to  be  left  in  this;  the  other  chamber 


124  A  CHEMICAL  SIGN  OF  LIFE 

is  for  the  purpose  of  quantitative  determination,  and  its 
stopper  need  not  be  removed.  The  tissue  is  carefully 
laid  on  the  glass  plate  and  on  the  platinum  electrodes 
fused  into  the  chamber,  or  it  can  be  laid  on  the  cover- 
slide  and  placed  on  the  mercury.  Close  the  stopper  R 
and  seal  both  chambers  with  mercury.  Wash  the 
apparatus  with  air  free  of  carbon  dioxide,  as  directed 
before.  At  the  end  of  the  sixth  or  seventh  washing 
stopcocks  G  and  F  are  closed  and  the  time  is  recorded, 
since  it  is  plain  that  from  this  time  on  we  are  retaining 
any  gas  given  off  by  the  tissue  in  the  chamber.  The 
apparatus  is  filled  once  more  with  air  free  of  carbon 
dioxide  by  opening  stopcock  7;  the  pressure  is  quickly 
adjusted  by  raising  the  safety  bottle  Z>,  while  the  stop- 
cock /  is  still  open,  and  then  /  is  shut.  After  opening 
stopcock  C,  barium  hydroxide  is  introduced  into  the 
tube  d  of  the  left  chamber  A  only,  but  the  solution  is 
never  introduced  into  the  respiratory  chamber  B.  Turn 
the  stopcock  L  in  such  a  way  as  to  sever  the  connection 
between  these  two  chambers.  It  is  imperative,  not  only 
that  the  hemispherical  drop  formed  at  d  in  the  left 
chamber  should  be  perfectly  clear  at  the  time  of  intro- 
duction of  this  solution,  but  also  that  no  visible  granule 
of  any  kind  should  be  produced  on  standing.  No 
quantitative  experiment  can  be  performed  unless  the  air 
is  absolutely  free  from  carbon  dioxide.  We  have  thus 
a  control  for  each  quantitative  experiment.  If  at  the 
end  of  the  desired  period  of  respiration,  say  ten  minutes, 
the  drop  is  perfectly  clear,  not  having  any  deposit  visible 
with  a  lens,  a  portion  of  the  gas  from  the  respiratory 
chamber  B  is  introduced  into  the  left  chamber.  This  is 
accomplished  by  drawing  a  designated  amount  of  mer- 


THE  BIOMETER:    HOW  TO  USE  IT  125 

cufy  from  the  left  chamber  A  by  opening  the  stopcock  C 
and  returning  the  same  amount  of  mercury  to  the  mer- 
cury burette  T,  opening  the  stopcock  Z,,  and  quickly 
shutting  the  stopcock,  so  that  the  communication  of 
the  gases  between  these  chambers  is  momentary.  This 
process  of  driving  the  known  amount  of  the  gas  from  the 
respiratory  chamber  to  the  analytic  chamber  must  be 
done  in  a  few  seconds.  The  volume  of  mercury  with- 
drawn from  the  analytic  chamber  is  easily  determined 
by  drawing  it  into  a  small  graduated  cylinder,  or,  more 
accurately,  by  weighing  it,  and  this  volume  corresponds 
to  the  exact  amount  of  the  gas  we  took  from  the  right 
chamber  to  the  left,  since  the  pressures  in  A  and  B  are 
kept  exactly  equal  to  atmospheric  pressure  during  the 
transfer  of  the  gas. 

One  now  watches  the  surface  of  the  drop  at  d  with  a 
lens  to  see  whether  or  not  any  deposit  is  formed  during 
ten  minutes.  The  presence  or  absence  of  any  visible 
precipitate  will  decide  whether  the  amount  of  gas 
taken  from  the  respiratory  chamber  contained  enough 
carbon  dioxide  to  give  a  visible  deposit.  With  this 
apparatus  we  have  repeatedly  introduced  accurately 
known  quantities  of  carbon  dioxide  of  very  high  dilution 
into  the  left  chamber  and  found  with  remarkable  regu- 
larity that  i.oXio"7  g.  of  carbon  dioxide  is  the  mini- 
mum amount  which  will  cause  a  formation  of  detectible 
precipitate  of  barium  carbonate  during  ten  minutes. 
Smaller  amounts  of  the  gas  than  this  will  give  no  pre- 
cipitate for  a  long  time,  while  larger  amounts  give  it  more 
quickly  and  it  appears  in  larger  quantities.  There  is  a 
sharp  line  of  demarkation  at  i .  oX  io~7  g.,  no  matter  how 
large  a  space  this  amount  of  gas  is  occupying  with  the  air. 


126  A  CHEMICAL  SIGN  OF  LIFE 

Thus  in  order  to  determine  the  concentration  of 
carbon  dioxide  in  question,  one  must  first  determine  how 
many  cubic  centimeters  of  the  gas  must  be  introduced 
before  we  can  obtain  the  precipitate  in  ten  minutes; 
this  volume  must  contain  then  i.oXio"7  g.  Since  we 
know  the  volume  of  the  original  respiratory  chamber 
from  which  this  known  amount  of  gas  is  withdrawn, 
we  can  easily  determine  how  much  total  carbon  dioxide 
is  present  at  the  time  of  analysis.  That  is  to  say,  the 
original  capacity  of  chamber  B  divided  by  this  mini- 
mum quantity  of  the  gas  which  gave  the  precipitate, 
multiplied  by  i.oXio"7  g.,  corresponds  to  the  total 
amount  of  the  carbon  dioxide  given  by  the  known  weight 
of  the  tissue  for  the  known  period  of  time. 

The  following  example  will  make  the  details  of  the 
method  and  calculation  clear: 

We  took  10  mg.  of  the  sciatic  nerve  of  a  frog  and 
after  ten  minutes  of  respiration  we  drew  i  c.c.  from  the 
respiratory  chamber  into  the  left  chamber,  and  found  no 
precipitate  visible  within  ten  to  fifteen  minutes.  Instead 
of  now  taking  more  gas  from  the  respiratory  chamber,  we 
should  take  another  fresh  nerve,  and,  after  it  has  respired 
ten  minutes  or  longer,  draw,  say,  1.5  c.c.  to  the  analytic 
chamber.  As  will  be  noticed,  we  have  three  variables 
which  we  can  choose  from,  namely,  the  weight  of  the 
nerve,  the  time  of  respiration,  or  the  amount  of  gas 
withdrawn  from  the  respiratory  chamber  at  the  time  of 
analysis.  To  estimate  the  carbon  dioxide  production 
from  the  isolated  tissues  it  is  far  better  to  keep  the  time 
constant  and  vary  the  other  two,  for  in  many  cases  the 
rate  of  respiration  varies  as  the  time  elapses.  As  far 
as  the  weight  of  the  tissue  is  concerned,  we  cannot  but 


THE  BIOMETER:    HOW  TO  USE  IT  127 

vary  it,  for  it  is  not  only  a  waste  of  time  to  try  to  get 
exactly  the  same  amount  of  the  tissue  for  each  experi- 
ment, but  in  many  cases  such  an  attempt  will  lead  to 
a  number  of  physiological  errors.  Of  course  there  is  a 
time  when  we  must  select  the  same  weights  of  the  tissues 
for  a  particular  experiment,  such,  for  instance,  as  when 
we  are  to  test  the  relation  of  the  sizes  of  the  tissue  and 
rate  of  the  carbon  dioxide  production. 

The  quantitative  experiments,  therefore,  consist  in 
determining  the  least  volume  of  the  gas  necessary  to  give 
the  precipitate  for  a  known  weight  of  the  tissue  for  a 
known  period  of  time.  This  can  be  found  by  experi- 
menting on  several  tissues  of  different  weights  (too  much 
variation  of  the  weight  should  be  avoided),  i.e.,  by 
obtaining  two  sets  of  results,  namely,  the  one  which  does 
not  give  the  precipitate  and  the  other  which  gives  the 
precipitate. 

These  results  are  calculated  on  the  standard  unit,  so 
that  we  can  compare  them  with  each  other.  We  have 
usually  taken  10  mg.  and  ten  minutes  as  units.  An 
example  will  explain:  14  mg.  of  the  nerve  for  fifteen 
minutes  of  respiration  did  not  give  a  precipitate  when 
we  took  but  i  c.c.  from  the  respiratory  chamber.  There- 
fore this  nerve  for  10  mg.  for  ten  minutes'  respiration 
will  not  give  any  precipitate  when  we  take  2 .  i  c.c.  from 
the  chamber.  In  another  case  we  took  13  mg.  of  the 
nerve,  which  after  ten  minutes'  respiration  produced  so 
much  carbon  dioxide  that  2  c.c.  gave  a  precipitate; 
thus  2 . 6  c.c.  will  give  precipitate  for  10  mg.  of  the  nerve 
for  ten  minutes'  respiration.  In  this  way  a  series  of 
experiments  with  several  fresh  nerves  was  conducted  in 
order  to  approximate  both  the  minimum  volume  which 


128 


A  CHEMICAL  SIGN  OF  LIFE 


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1                     5 

THE  BIOMETER:    HOW  TO  USE  IT  129 

will  precipitate  and  the  maximum  volume  which  does 
not  give  a  precipitate  for  a  definite  time  and  weight.  In 
Table  I  columns  8  and  9  refer  to  these  columns  calculated 
from  the  experiments  for  ten  minutes'  respiration  by 
10  mg.  of  the  nerve.  Since  we  know  that  the  minimum 
volume  which  gave  a  precipitate  must  contain  a  definite 
amount  of  carbon  dioxide,  i.e.,  i.oXio"7  g.,  and  since 
we  had  15  c;c.  of  original  volume  of  the  respiratory  cham- 
ber, we  can  calculate  the  total  amount  of  the  gas  given 
off  by  the  sciatic  nerve  of  the  frog. 

APPARATUS  III 

Although  the  use  of  the  biometer  is  perfectly  satis- 
factory for  almost  all  micro-metabolic  analyses,  and 
sometimes  is  indispensable  for  a  quick 'quantitative  com- 
parison of  two  different  rates  of  carbon  dioxide  production 
from  the  different  tissues,  yet  it  is  extremely  inconvenient 
for  a  complete  determination  of  carbon  dioxide  pro- 
duction from  a  single  tissue,  the  metabolic  rate  of  which 
is  constantly  changing  and  the  availability  of  which  is 
not  very  great.  The  necessity  for  a  new  device  to  meet 
this  difficulty  was  keenly  felt  when  we  were  studying  the 
metabolic  changes  before,  during,  and  after  the  cleavage 
of  a  single  fish  egg. 

The  new  feature  of  this  special  apparatus  is  a  device 
by  which  the  air  after  a  definite  period  of  respiration  by 
the  tissue  can  be  withdrawn  into  a  tube  from  the  respira- 
tory chamber  for  subsequent  complete  analysis.  With 
the  new  arrangement,  therefore,  one  can  make  not  only  a 
complete  analysis  with  a  single  tissue,  but  also  several 
duplicate  determinations. 


130  A  CHEMICAL  SIGN  OF  LIFE 

Description  of  Apparatus  III. — As  shown  in  Fig.  5, 
the  main  part  of  this  apparatus  consists  of  only  one  glass 
bulb  A,  which  serves  the  combined  purposes  of  respira- 
tory and  analytical  chambers  of  the  biometer.  Its 
capacity  is  about  30  to  40  c.c.,  but  can  become  smaller 
by  introducing  mercury  in  the  same  way  as  we  managed 
in  the  other  apparatus.  The  barium  hydroxide  tube  d 
is  inserted  through  its  wall,  and  the  three-way  stopcock  4 
is  attached  to  the  bottom  of  the  chamber.  Just  opposite 
the  top  of  the  barium  hydroxide  tube  d  there  is  another 
three-way  stopcock  2,  one  arm  of  which  is  connected  to 
the  nitrometer  C  and  the  other  arm  of  which  is  con- 
nected to  tube  B,  into  which  the  respired  air  is  to  be 
drawn  for  a  subsequent  analysis.  This  tube  B  is 
attached  to  a  mercury  burette  G,  by  which  the  pressure 
in  the  tube  and  the  chamber  can  be  adjusted.  The 
similar  mercury  burette  H  is  attached  to  the  chamber 
proper  for  the  same  purpose  as  well  as  for  the  means  of 
driving  air  into  the  tube  B.  The  remaining  parts  of  the 
apparatus  are  exactly  the  same  as  in  the  biometer  and 
are  shown  in  the  figure  with  dimensions. 

Method  for  quantitative  determination  of  carbon  dioxide 
with  apparatus  III. — The  detailed  method  is  as  follows : 
Open  stopcocks  2  and  3  in  such  a  way  as  to  connect  the 
chamber  A  and  the  tube  B  only.  Fill  the  tube  B  with 
mercury  by  raising  the  mercury  burette  G.  Close  stop- 
cock 2  when  a  little  excess  of  mercury  is  pushed  over  into 
the  space  in  the  capillary  tube  between  the  chamber  A 
and  the  tube  B  and  when  the  tube  B  is  known  to  be 
absolutely  free  from  any  bubble  of  air.  The  closing  of 
the  stopcock  must  be  done  in  such  a  way  that  there 
is  a  connection  made  between  the  chamber  A  and 


THE  BIOMETER:    HOW  TO  USE  IT 


132  A  CHEMICAL  SIGN  OF  LIFE 

the  nitrometer  C.  Increase  the  pressure  inside  of  the 
nitrometer  C  by  raising  the  safety  bottle  D  above  the 
level  of  the  alkali  in  the  nitrometer,  and  then  open 
stopcock  /.  In  this  way  the  excess  of  mercury  left 
in  the  capillary  tube  will  be  pushed  over  into  the 
chamber  A  and  flow  through  stopcock  4  into  a  receiv- 
ing vessel. 

If  stopcocks  2  and  3  are  absolutely  air-tight,  there 
should  be  no  air  bubble  present  in  the  tube  B  on  standing. 
This  being  assured,  a  known  amount  of  mercury  is  intro- 
duced into  the  chamber  A  by  means  of  mercury  burette 
H,  thus  making  the  capacity  of  the  chamber  what  was 
desired.  Tissue  is  introduced  into  the  chamber  in  the 
usual  manner,  the  glass  stopper  is  replaced,  the  chamber 
is  sealed  with  mercury,  and  the  nitrometer  is  filled  with 
air  free  from  carbon  dioxide.  After  evacuation  of  the 
chamber  and  introducing  pure  air  several  times,  stop- 
cock 5  is  closed  and  the  time  is  recorded,  the  pressure  is 
adjusted,  and  stopcock  2  is  turned  45°.  At  the  end  of 
the  desired  period  of  respiration  any  portion  of  the  air, 
say  10  or  15  c.c.,  from  the  chamber  can  be  driven  into 
tube  B.  This  is  accomplished  by  raising  the  right-hand 
mercury  burette  H  and  by  simultaneously  opening 
stopcocks  2  and  4  and  gradually  lowering  the  left-hand 
mercury  burette  G.  Stopcock  2  is  now  closed  and  the 
pressure  of  the  air  in  B  is  made  equal  to  that  of  the 
atmosphere  and  is  kept  under  this  condition,  having 
the  mercury  burette  G  at  the  proper  height. 

Remove  mercury  from  the  stopper  S  and  unstop  the 
chamber,  take  away  the  tissue,  and  lower  the  mercury 
burette  H  so  that  all  the  mercury  in  the  chamber  will 
flow  back  into  the  burette.  The  little  excess  of  mercury 


THE  BIOMETER:    HOW  TO  USE  IT  133 

now  left  in  the  chamber  A  can  be  withdrawn  through 
stopcock  4  into  a  receiving  vessel.  In  order  now  to 
analyze  the  air  in  the  tube  B,  it  is  better  to  clean  the 
apparatus  once  more  with  water  and  dry  it,  as  directed 
elsewhere. 

The  chamber  is  now  filled  with  mercury  so  that  the 
remaining  volume  of  it  will  be  as  little  as  possible,  say 
15  c.c.  (the  exact  volume  need  not  be  known  here), 
the  apparatus  is  sealed  with  mercury  as  usual,  and  then 
washed  several  times  with  air  free  of  carbon  dioxide,  and 
then  clear  barium  hydroxide  is  introduced  into  the  usual 
tube  inside  of  the  chamber,  forming  a  hemispherical 
drop  at  the  top  of  d.  If  no  deposit  of  barium  carbonate 
forms  on  the  surface  of  the  drop  within  ten  or  fifteen 
minutes,  we  are  sure  that  ordinarily  the  air  we  use  is 
free  from  carbon  dioxide  and  that  the  apparatus  is  in 
perfect  condition.  This  point  established,  a  small 
portion  of  the  gas  is  driven  from  the  tube  B  into  this 
chamber  A.  This  is  done  by  withdrawing  a  desired 
amount  of  mercury  from  the  chamber  A  into  a  receiving 
cylinder  and  adjusting  the  pressure  in  the  chamber  and 
tube  B  by  means  of  mercury  burette  G.  Close  stopcock 
2  by  turning  it  45°. 

The  surface  of  the  drop  at  d  should  now  be  watched 
with  a  lens,  as  usual,  for  a  deposit  of  carbonate.  If  no 
deposit  appears  within  ten  minutes,  we  should  introduce 
more  air  from  the  tube,  with  usual  care,  until  we  get  the 
first  visible  precipitate  detectible  with  a  lens  during  ten 
minutes'  standing.  It  is  very  important  that  we  should 
give  about  ten  minutes  of  time  for  the  reaction  after 
each  withdrawal  of  the  air  from  the  tube  B  into  the 
chamber  A . 


134  A  CHEMICAL  SIGN  OF  LIFE 

The  following  calculation  will  make  the  method 
clearer : 

The  original  volume  of  the  respiratory  chamber  is 
31.4  c.c.,  to  which  6.4  c.c.  of  mercury  are  introduced, 
making  the  remaining  volume  exactly  25  c.c.  Ten 
milligrams  of  the  tissue  are  used  and  are  allowed  to 
respire  in  the  chamber  for  ten  minutes.  Then  about 
10  to  15  c.c.  of  the  gas  are  withdrawn  into  the 
tube  B;  0.5  c.c.  of  this  gas  gave  no  precipitate 
during  the  first  ten  minutes;  0.5  c.c.  more  of  the 
same  sample  gave  no  deposit  in  another  interval  of 
ten  minutes.  Thereupon  0.5  c.c.  more,  a  total  of 
1.5  c.c.,  was  run  into  the  chamber.  A  marked  evi- 
dence of  a  precipitate  appeared  in  ten  minutes.  There- 
fore i .  5  c.c.  of  this  gas  must  contain  i .  oX  io~7  g.  of 
carbon  dioxide. 

The  apparatus  is  then  cleaned  and  dried  and  a  clear 
drop  of  barium  hydroxide  is  again  introduced  upon  the 
top  of  the  tube  d;  and  after  making  sure  that  the  air  is 
free  from  any  carbon  dioxide  by  waiting,  i  c.c.  of  the 
sample  gas  which  has  been  left  undisturbed  in  the  tube  B 
is  introduced  into  the  chamber;  no  precipitate  will  be 
found  to  have  formed  within  ten  minutes;  0.25  c.c. 
more  of  the  sample  will  not  produce  any  precipitate; 
but  if  0.25  c.c.  more  is  taken,  crystals  of  barium  car- 
bonate appear  after  ten  minutes.  It  follows  that  i .  5  c.c. 
of  the  respired  gas  must  contain  i.oXio"7  g.  of  carbon 
dioxide. 

From  these  duplicates  it  becomes  certain  that  i .  5  c.c. 
of  25  c.c.  capacity  of  the  chamber  now  contain  i.oX 
io~7  g.  of  carbon  dioxide.  Therefore  the  total  amount 


THE  BIOMETER:    HOW  TO  USE  IT  135 

of  carbon  dioxide  produced  by  10  mg.  of  the  tissue 
during  ten  minutes  will  be 

i .  oX  io-7  g.X— =  16 .  6X 10-7  g. 

of  carbon  dioxide. 

In  order  to  test  the  accuracy  with  which  our  new 
method  can  be  used  for  the  estimation  of  the  exceedingly 
minute  quantities  of  the  carbon  dioxide,  a  series  of 
determinations  was  made  on  the  samples  whose  con- 
centrations were  unknown  to  the  experimenters  at  the 
time  of  analysis. 

The  results  are  given  in  Table  II : 

TABLE  II 


VOLUME  OP  SAMPLE 

WEIGHT  OF  ( 

rOa  IN  I  C.C. 

REQUIRED  TO  GIVE  A 
PRECIPITATE 

Found 

Taken 

I   O     C  C.. 

i  o  Xio    7  g. 

O.Q2XlO~7g. 

0.5    c.c  

2.0  Xio~7g. 

2.3    Xio~-7g. 

o  55  c.c. 

i  82Xio~7g. 

i.83Xio~7g. 

i  .  5    c.c  

o.67Xio~~7g. 

o.62Xio~7  g. 

2    25  C  C. 

o  45X10    7  g. 

o.45Xio~7g. 

One  disadvantage  of  this  apparatus  III  is  that  we 
must  take  into  consideration  temperature  and  pressure 
variation,  which  was  entirely  unnecessary  for  the 
biometer  proper.  If  the  respiration  and  analysis  are 
carried  out  at  different  temperature  and  pressure, 
the  ratio  between  the  minimum  volume  which  gives 
the  first  precipitate  and  the  original  volume  of  the 
chamber  will  not  be  rigid.  In  that  case  the  minimum 
volume  should  be  translated  to  the  volume  at  the 
temperature  and  pressure  at  the  time  of  respiration. 
Such  correction,  however,  will  not  be  necessary  if  the 


136  A  CHEMICAL  SIGN  OF  LIFE 

analysis  is  done  immediately  after  the  respiration,  dur- 
ing which  the  variation  in  temperature  and  pressure 
will  not  affect  the  results  beyond  experimental  errors, 
as  is  shown  in  the  following  calculation: 

Let  us  suppose  that  10  mg.  of  the  tissue  respire  for  ten 
minutes  at  18°  C.  under  760  mm.  of  pressure  in  25  c.c. 
of  the  chamber,  and  suppose  i  .  5  c.c.  of  the  air  at  22°  C. 
and  730  mm.  of  pressure  gave  the  first  precipitate.  We 
shall  then  obtain  the  following  results: 

a)  Without  correction  we  get 

i.  oX  io-7  g.X  —  =  16.6X10-7  g. 

b)  With  a  correction, 


(270+  2  2)  X  760 

This  shows  a  little  over  5  per  cent  error,  which  will  be 
the  maximum  and  almost  an  impossible  variation,  con- 
sidering the  ordinary  weather  in  the  laboratory  for  a 
short  interval  of  time.  Besides,  we  are  dealing  with  a 
very  small  sample  of  moist  tissue,  the  weight  of  which 
may  easily  vary  within  5  per  cent. 


INDEX 


NOTE. — References  give  the  number  of  the  page  on  which  the  matter  referred  to 
begins. 


Acid,  23;  production  in  eggs,  41; 
in  muscle,  41;  in  nerves,  25. 

Action  current,  13. 

Aerobic  tissue,  15. 

Afferent  fiber,  58. 

Air,  pure,  23. 

Algae,  red,  92. 

Alkali,  Greenbank,  114. 

Anaerobic  tissue,  15. 

Anesthesia:  partial,  on  local  exci- 
tability, 58;  on  conductivity, 
58.  See  also  Carbon  dioxide 
production. 

Anesthetics,  25,  61;  on  refractory 
period,  48.  On  carbon  dioxide 
production  see  Carbon  dioxide. 
See  also  Ether,  Urethane, 
Chloral  hydrate. 

Animal  heat:  discovery  of  nature 
of,  n;  source  of,  n,  51. 

Apparatus,  for  carbon  dioxide 
determination.  See  Biometer; 
Apparatus  III. 

Apparatus  III,  129;  description, 
130;  diagram,  131;  method, 
130. 

Arachnid,  So. 

Arbacia,  41. 

Asphyxiation:  on  refractory 
period,  48;  as  cause  of  anes- 
thesia, 60. 

Astacus,  30. 

Atmospheric  oxygen.    See  Oxygen. 

Automatic  ganglion.  See  Gan- 
glion. 

Axis  cylinder,  50,  51. 

Bacterial  decomposition,  27,  28. 
Banana  fly,  no. 


Barium  carbonate:  detection  of, 
in;  precipitation  of,  16,  19; 
solubility  of,  15;  stages  of 
granulation  of,  diagrams,  123. 

Barium  hydroxide,  15,  19,  20,  89, 
94,  no,  114,115,  118,  121,  130, 
133- 

Bayliss,  29,  33,  45,  note  i,  18. 

Bicycle,  compared  to  life-process, 
107. 

Biometer,  6,  15,  17,  19;  accuracy 
of,  135;  bubbles  in,  123;  cal- 
culating results,  127,  128;  cali- 
brating volume  of,  118;  carbon 
dioxide,  free  air  for,  116;  clean- 
ing of,  115;  description  of,  in; 
diagram  of,  113;  photograph  of, 
17,  112;  principles  of,  no; 
qualitative  use  of,  120;  quanti- 
tative use  of ,  1 23 ;  quick  quanti- 
tative comparison  with,  122; 
sensitiveness  of,  in;  setting 
up,  114;  testing  purity  of  air 
in,  117;  uses  of,  109. 

Blaze  current,  5,  88,  101.  See 
also  Electrical  signs  of  life. 

Blood  supply,  16. 

Brain,  16,  59;  increased  metab- 
olism in,  36;  ring,  76. 

Brown,  Horace,  87. 

Burch,  47. 

Cancer,  pagurus.  See  Carbon 
dioxide  production. 

Carbohydrate,  10. 

Carbon  dioxide  production:  by 
life  process,  22;  comparison  of, 
in  nerve  and  other  tissues,  29; 
in  different  animals:  crabs, 
cancer  pagurus,  30;  crayfish, 
astacus,  30;  Crustacea,  30;  dog, 


137 


138 


A  CHEMICAL  SIGN  OF  LIFE 


30;  frogs,  Rana  esculenta,  and 
temporaria,  30;  in  ganglion, 
Limulus  polyphemus,  32;  in 
muscle,  frog,  Rana  temporaria, 
30;  lobster,  Homarus  vulgaris, 
30;  man  at  rest,  30;  in  differ- 
ent parts  of  nerve,  76,  77;  in 
hydrogen,  26,  43;  in  "inexci- 
table"  nerves,  65;  in  killed 
nerves,  24;  in  nerves:  carp, 
R.  lat.  rag.,  and  R.  lat.  ace.,  79; 
catfish,  R.  lat.  vag.,  and  R.  lat. 
ace.,  79;  chloral  hydrate,  65; 
dog,  anterior  root,  posterior 
root,  hypoglossal,  79;  frog, 
Rana  pipiens,  sciatic,  vesting, 
and  stimulated,  32;  guinea 
pig,  22;  hypoglossal,  79;  "in- 
excitable,"  62;  Limulus,  claw 
nerve,  32;  mouse,  22;  optic 
nerve  (whole)  proximal  and  dis- 
tal, 32;  rabbit,  22;  rat,  22; 
6  skate,  Raia  erinecia,  and 
Raia  ocallata,  optical,  olfactory, 
oculomotor,  22;  spider  crab, 
Libinia  canaliculata,  claw  nerve, 
whole,  proximal,  distal,  32; 
squiteague,  cy  no  scion  ^  regalis, 
22;  stimulated,  non-stimulated, 
under  treatment  of  different 
concentrations  of  ethyl  ure- 
thane,  62;  turtle,  22;  under 
anesthesia,  25 ;  in  resting  nerves, 
19,  22,  32;  in  stimulated  nerves, 
at  successive  time  intervals,  65; 
under  anesthesia,  25,  61. 

Carbon  dioxide:  as  a  measure  of 
metabolism,  12;  as  end  product 
of  metabolism,  n,  34;  gradient, 
79;  increment  of,  on  stimula- 
tion as  sign  of  life,  87,  chap,  v; 
influence  on  electrical  change, 
14;  method  of  analysis  of,  see 
Biometer;  method  of  detecting, 
in  nerve,  16,  20;  sources  of, 
23- 

Carbon  dioxide  free  air,  116. 

Carbonate,  23. 

Carp.  See  Carbon  dioxide  pro- 
duction. 


Cat  fish.  See  Carbon  dioxide 
production. 

Chemical  energy,  84. 

Chemical  processes,  various,  in 
the  living  matter,  10. 

Chemical  sign  of  life,  algae,  93;  in 
Australian  pine,  92;  in  common 
glass,  92;  in  corn,  91;  in 
Japanese  ivy,  9  2 ;  in  Lincoln  oats , 
91;  in  mustard  seeds,  91;  in 
nerve,  34,  55;  in  rice,  91; 
in  Swedish  selected  oats,  91;  in 
wheat,  89. 

Chemical  stimulation.  See  Stim- 
ulation. 

Chemical  transformation,  12. 

Child,  74,  81. 

Chloral  hydrate,  61.  See  also 
Carbon  dioxide  production. 

Chloroform,  61. 
Chlorophyll,  2. 
Claude  Barnard,  48. 
Claw  nerve.     See  Nerve. 
Cold-blooded  animals,  22. 
Conducting  medium,  18. 
Conduction:    as  phenomenon   of 
living  matter  4;    also  chap.  iv. 

Conductivity,  6.  See  also  Con- 
duction. 

Connective  tissue,  16,  18,  46. 

Contractility,  8. 

Crab.    See  Cancer  pagurus. 

Crayfish.    See  Astacus. 

Crocker,  92. 

Crustacea.  See  Carbon  dioxide 
production. 

Current  of  action.  See  Action 
current. 

Cyanide.     See  Potassium  cyanide. 

Cynosion  regalis.  See  Carbon  di- 
oxide production. 

Daniel  cell,  52. 
Death,  rapidity  of,  73. 


INDEX 


139 


Dendrite,  77;    metabolic  gradient 

in  sensory,  78. 
Dextrose,  82. 
Diphasic  current,  13. 
Dog,  metabolism  in.     See  Carbon 

dioxide  production. 
Drugs,  on  refractory  period,  48. 
Dry  seed.    See  Seed. 
Dyer,  Thistleton,  87. 

Efferent  fiber,  58;  gradient  in, 
see  Metabolic  gradient. 

Eggs,  production  of  acid  in,  41. 

Ehrlich,  37. 

Electrical  changes:  as  functional 
change,  4;  discovery  of,  4; 
as  sign  of  passage  of  nerve  im- 
pulse, 23;  influence  of  carbon 
dioxide  on,  13;  See  also  Blaze 
current;  Electrical  sign  of  life. 

Electrical  current,  37. 

Electrical  resistance,  changes  in, 
after  stimulation,  101. 

Electrical  sign  of  life.  See  Blaze 
current;  Electrical  resistance. 

Electricity:  measure  of,  3;  ve- 
locity of,  8. 

Electrodes,  12,  13,  40. 

Electromotive  force,  in  nerve,  35. 

Electropositive,  58. 

Energy,  source  of,  in  living  matter, 
9,  ii. 

Ether,  25,  61;  for  effect  of,  on 
carbon  dioxide  production,  see 
Carbon  dioxide  production. 

Ethyl  ure thane,  25,  63.  See  also 
Carbon  dioxide  production. 

Excitability:  degree  of,  59;  de- 
pression of,  see  Anesthetics; 
its  relation  to  conductivity,  57, 
chap,  iv;  its  relation  to  metab- 
olism, 59;  three  criteria  of, 
59 ;  transmission  of ,  41 .  See  also 
Irritability. 

Faraday,  3. 
Fat,  10. 


Fatigue:  as  metabolic  sign,  49; 
lack  of,  in  nerve,  8,  46. 

Fermentation,  10,  23,  25,  27. 

Film,  of  barium  hydroxide,  15,  16. 

Fletcher,  30,  41. 

Forgetting,  phenomenon  of,  35. 

Frog.  See  Carbon  dioxide  pro- 
duction. 

Frohlich,  45. 

Functional  changes,  4;  in  the 
nerve  impulse,  7;  invisible,  4; 
See  also  Electrical  changes; 
Chemical  changes. 

Fundulus  hectroclitus,  no. 

Galen,  n. 

Galvani,  5,  13. 

Galvanometer,  3,  5,  13,  23. 

Ganglion,  31;  comparison  with 
metabolism  of  nerve  fiber,  31; 
heart  ganglion,  32.  See  also 
Carbon  dioxide  production. 

Glass  plate,  20,  37;   diagram,  38. 

Gotch  and  Burch,  47. 

Gradient:  metabolic,  79;  in 
afferent  fiber,  76;  in  efferent 
fiber,  72;  in  sensory  dendrite, 
77;  relation  of,  to  direction  of 
impulse,  78,  80;  structural,  75. 

Green  pigment,  2. 

Growth,  10,  35. 

Guinea-pig.  See  Carbon  dioxide 
production. 

Haberlandt,  54. 

Heart,  31. 

Heat  coefficient,  52. 

Heat  formation:   in  brain,  36;   in 

nerve    7,    49;    its    relation    to 

metabolism,  5. 
Helmholtz,  50. 
Herrick,  71. 

Hill,  30;  on  heat  formation,  50. 
Homarus    vulgaris.    See    Carbon 

dioxide  production. 
Hopkins,  41. 


140 


A  CHEMICAL  SIGN  OF  LIFE 


Horseshoe  crab.  See  Limulus 
polyphemus. 

Hydrogen,  26,  60;  decreased 
metabolism  in  44;  lack  of  in- 
creased metabolism  in,  44. 

Hydrolysis,  52. 

Hypoglossal  nerve.    See  Nerve. 

Impulse:  in  plant,  2;  nerve,  see 
Nerve  impulse. 

Inhibition:  by  heat,  58;  non- 
transmissibility  of,  58. 

Injury,  41,  91. 

Insect,  wings  of,  47. 

Invertebrate,  18. 

Irritable  response,  103. 

Irritability:  definition  of,  4; 
metabolic  condition  for,  85; 
nature  of,  104;  origin  of  physi- 
cal theory  of,  9;  relation  of,  to 
conductivity,  57;  relation  of,  to 
metabolism,  104;  two  phases  in 
protoplasmic,  57. 

Katabolism,  48. 

King  crab.  See  Limulus  poly- 
phemus. 

Lateral  line  nerve.    See  Nave. 

Lavoisier,  n. 

Learning,  phenomenon  of,  35. 

Lens,  15,  16. 

Libinia  canaliculata.    See  Carbon 

dioxide  production. 
Life:    chemical  sign  of,  chap,  v; 

definition  of,  95;    quantity  of, 

107. 

Light,  35. 
Limax,  22. 

Limulus.  See  Limulus  polyphemus . 
Limulus  polyphemus.    See  Carbon 

dioxide  production. 
Living    process,     103;      material 

changes  in,  3;  physical  state  of, 

105. 
Living  things :  property  of,  3;  two 

signs  of,  4. 
Lobster.    See  Homarus  vulgar  is. 


Man.  See  Carbon  dioxide  pro- 
duction. 

Mathews,  35,  41. 

Mayer,  82. 

Mayow,  n. 

Medullary  sheath,  51,  58;  func- 
tion of,  18. 

Medullated  nerve,  18,  21.  See 
also  Carbon  dioxide  production. 

Memory,  35. 

Metabolic  gradient.  See  Gra- 
dient. 

Metabolism:  function  of,  85,  104; 
increased,  on  stimulation,  34; 
in  different  tissues  and  organ- 
isms, see  Carbon  dioxide  pro- 
duction; indirect  evidence  for, 
in  nerve,  12;  its  meaning,  50, 
104;  method  of  study  of,  in 
nerve,  see  Biometer;  relation  to 
behavior,  81;  resting,  33,  89. 

Methylene  blue,  37. 

Motor  nerve.     See  Nerve. 

Mouse,  22. 

Muscle:  acid  production  in,  91; 
contraction  of,  as  sign  of  nerve 
impulse,  23;  increased  metab- 
olism of  contracting,  35; 
smooth,  51;  voluntary,  18. 

Narcosis.    See  Anesthesia. 
Narcotics.    See  Anesthetics. 
Negative  phase,  13. 
Negative    response,    influence    of 
carbon  dioxide,  14. 

Nerve:  afferent,  58;  claw,  18; 
efferent,  58;  hypoglossal,  79; 
lateral  line,  22;  motor,  21; 
oculomotor,  21;  olfactory,  21; 
optic,  22;  sciatic,  21,  22,  67; 
sensory,  22,  58;  nerves  of 
different  animals.  See  also 
Carbon  dioxide  production. 

Nerve  fibers:  Changes  of  weight 
under  anesthesia,  62,  65;  chem- 
ical change  in,  chaps,  ii  and  iii; 
chemical  sign  of,  5^;  different 


INDEX 


141 


'kinds  of,  58;  electrical  changes 
in,  5,  12,  13;  function  of,  6; 
functional  changes  in,  7;  heat 
formation  in,  7,  50;  lack  of 
visible  sign  of  vitality  in,  7; 
longevity  of  isolated,  24;  meta- 
bolic gradient  in,  72;  metabol- 
ism on  stimulation,  55;  oxygen 
consumption  by,  53;  physical 
changes  in,  7;  production  of 
acid  in,  25;  property  of,  6; 
resting  metabolism  in,  22; 
structural  changes,  7;  struc- 
tural gradient  in,  75;  use  of 
isolated,  23. 

Nerve  impulse,  6;  direction  of, 
72;  direction  of,  and  metab- 
olic gradient,  77;  effect  of 
salt  on,  82;  effect  of  tempera- 
ture on,  83;  nature  of,  85; 
nature  of  material  basis  of,  167; 
velocity  of,  8;  velocity  of,  and 
resting  metabolism,  80. 

Nitrogen,  electrical  response  in, 
45- 

Non-medullated  nerve,  18. 

Nucleus,  cell,  on  oxidation,  16. 

Oculomotor  nerve.    See  Nerve. 

Olfactory  nerve.     See  Nerve. 

Optic  nerve.     See  Nerve. 

Organic  compounds,  22. 

Osterhout,  92. 

Oxidation :  measure  of,  see  Metab- 
olism; r61e  of  nucleus  on,  16. 

Oxygen:  as  essential  to  life,  n; 
atmospheric,  15;  consumption 
by  brain,  36;  consumption  by 
linseed  oil,  35;  consumption  by 
nerve  fibers,  53;  consumption 
by  stimulated  nerve,  50,  53; 
consumption  under  anesthesia, 
60;  deficiency  of,  on  nerve  me- 
tabolism, 27;  discovery  of ,  n; 
r61e  of,  on  excitability,  54,  60; 
role  of,  in  salt  stimulation,  26. 

Painting,  chemical  process  in,  34. 
Peas,  31. 


Permeability,  81,  103. 

Plants:  differentiation  from  ani- 
mals, 2;  electrical  response  in, 
5;  resemblance  to  animals,  2; 
response  in,  2.  See  also  Seed. 

Pneumatic  spirit,  n. 

Positive  phase,  13. 

Positive  response,  influence  of 
carbon  dioxide  on,  14. 

Potassium  chloride,  42. 

Potassium  cyanide,  75. 

Priestly,  n. 

Propagation,  of  excitability.  See 
Nerve  impulse. 

Proteins,  10. 

Protoplasm,  48;  physical  condi- 
tion in,  8. 

Protoplasmic  respiration,  35. 

Psychic  change,  3. 

Psychic  life,  evolution  of,  2;  in 
child,  2;  in  seed,  3;  physical 
basis  of,  3. 

Psychic  process,  physical  process, 
accompanying,  4. 

Psychism,  3;   indirect  measure  of, 

6. 
Psychometer,  3. 

Rabbit.  See  Carbon  dioxide  pro- 
duction. 

Raid  erinecia.  See  Carbon  diox- 
ide production. 

Raia  ocallata.  See  Carbon  dioxide 
production. 

Ramus   later  alls   accessorius.    See 

Nerve. 
Ramus  later  alls  vagis.    See  Nerve. 

Rana  esculenta.  See  Carbon  di- 
oxide production. 

Rana  pipiens.  See  Carbon  dioxide 
production. 

Rana  temporaria.  See  Carbon 
dioxide  production. 

Rat.  See  Carbon  dioxide  pro- 
duction. 


142 


A  CHEMICAL  SIGN  OF  LIFE 


Red  pigment,  2. 

Refractory  period,  48;  absolute 
phase  of,  48;  conditions  affect- 
ing, 48;  relative  phase  of,  48. 

Respiration,  definition  of,  10; 
as  fundamental  to  vital  process, 
n. 

Respiratory  metabolism,  how  to 
study,  15. 

Resting  metabolism.  See  metab- 
olism. 

Riggs,  42. 

Ringer's  solution,  28. 

Rolleston,  50. 

Salts:  permeability  of,  to  nerve, 
8;  effects  on  nerve  impulse,  82; 
Ringer's  solution,  28;  sodium, 
on  metabolism,  43. 

Sciatic  nerve.    See  Nerve. 

Secretion,  8. 

Seeds:  blaze  current  in,  5,  88; 
chemical  signs  in,  88;  dete- 
rioration of,  87;  effect  of 
anesthetics  on,  91;  electrical 
sign  in,  5,  88;  increased  metab- 
olism of,  on  injury,  90; 
invisible  response  in,  4;  metab- 
olism in  killed,  01;  old  Egyp- 
tian, 88;  psychic  life  in,  3; 
vitality  of,  at  low  temperature, 
87. 

Sensory  dendrite.     See  Dendrite. 

Sensory  nerve.    See  Nerve. 

Sheath.    See  Medullary  sheath. 

Sisyphus,  84. 

Skate.    See  Raia. 

Smooth  muscle.    See  Muscle. 

Snyder,  51. 

Sodium  chloride,  42. 

Spider  Crab.  See  Carbon  dioxide 
production. 

Squiteague.  See  Carbon  dioxide 
production. 


Staircase,  14. 

Stewart,  50. 

Stimulation:  Chemical,  40;  effect 
of  repeated,  on  electrical  changes 
14;  electrical,  37;  injury  as,  91; 
mechanical,  41,  91;  other  stim- 
ulations, 39. 

Stomata,  92. 

Surface  tension,  8. 

Tait,  48. 

Turtle,  22. 

Temperature:     effect    on    nerve 

impulse,    82;     effect   on   nerve 

metabolism,  83. 
Thorner,  45. 
Transmission,  of  excitability.    See 

Nerve  impulse. 
Twelfth  dynasty,  88. 

Universe,  2. 

Unstable  equilibrium,  105. 

Urethane.    See  Ethyl  urethane. 

Velocity:  its  relation  to  resting 
metabolism,  80;  of  the  nerve 
impulse,  different  salt  concen- 
tration on,  82;  of  various  crus- 
tacean nerves,  81;  temperature 
on,  83. 

Vertebrates.  See  Carbon  dioxide 
production. 

Viscera,  nerve  to,  18. 

Voluntary  muscle.     See  Muscle. 

Waller,  u;  metabolism  of  nerve, 
12;  on  blaze  current,  5;  on 
changes  of  electrical  resistance, 
101;  on  longevity  of  nerve,  24; 
on  protoveratrin,  48;  on  stair- 
case, 13. 

Water,  as  end  product  of  metab- 
olism, ii. 

Weston  cell,  52. 

Yohimbin,  on  refractory  period, 
48. 


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